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Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy
Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 1209 –1217, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(98)00314-1
Original Contribution NONINVASIVE EVALUATION OF IN VIVO FREE RADICAL REACTIONS CATALYZED BY IRON USING IN VIVO ESR SPECTROSCOPY NOPPAWAN PHUMALA, TATSUO IDE,
and
HIDEO UTSUMI
Department of Biophysics, Faculty of Pharmaceutical Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan (Received 10 August 1998; Revised 26 October 1998; Accepted 26 October 1998)
Abstract—The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 mmol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that “chelatable iron” contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron. © 1999 Elsevier Science Inc. Keywords—Iron overload, In vivo ESR, Nitroxyl probe, Desferrioxamine, Free radicals
INTRODUCTION
actions [5– 8]. Because low molecular weight forms of iron have been detected in iron overload [9 –12] and other oxidative stress related conditions [13–16], this form of iron may play a catalytic role in free radical reactions. Although the spin trapping technique using X-band ESR provides information about species of free radicals in vitro [1,17,18], the formation of hydroxyl radicals by iron is controversial [1,19 –22]. Unfortunately, there are few reports that provide direct evidence of iron-induced free radical reactions in vivo. Recently, the technique of secondary radical spin trapping has been introduced to study free radical reactions in animal models. The detection of the DMSO-derived PBN/ • CH3 radical adduct in bile samples has provided indirect evidence of hydroxyl radical generation in iron-overloaded rats [23–25]. However, this technique is limited to reactions that presumably occur in the liver and excrete the spin adduct into the bile. A real time and non-invasive measurement of in vivo free radical reactions in iron overloaded animals is quite important to fulfill the information about association between catalytic activity and toxic form of iron in vivo, although spin-trapping technique clearly clarifies reactive oxygen species.
Iron is an essential element for biological functions in aerobic organisms, but there is growing evidence that implicates iron in several diseases, including cancer and arthritis [1,2]. Hemochromatosis is a classic example of iron toxicity. The massive iron overload in the vital organs, including the endocrine glands, heart, and liver, causes organ dysfunction and hepatic cirrhosis [3]. The major causes of death in inherited hemochromatosis patients are cardiac disease, liver failure, and hepatocellular carcinoma [4]. It has long been suspected that free radicals play a role in iron toxicity, because of their powerful oxidant activity, that has been demonstrated in in vitro experiments. Iron-induced lipid peroxidation and cellular injuries were prevented by antioxidants, presumably involving free radical reactions. Changes in the level of antioxidants and antioxidant enzymes imply ongoing free radical reAddress correspondence to: Hideo Utsumi, Department of Biophysics, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Tel: 181 92-642-6621; Fax: 181 92-642-6626; E-Mail: [email protected]. 1209
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In vivo ESR spectroscopy (L-band and 300 MHZ) has recently been developed to measure free radicals in living animals. Because its sensitivity relative to the amounts of endogenous free radical produced in vivo is poor, nitroxyl stable radicals (piperidine-N-oxyl and pyrrolidine-N-oxyl derivatives) have been used as spin probes for in vivo ESR measurement. The decay of nitroxyl ESR signals is influenced by the generation of active oxygen species [26], the biological redox system [27–29] and oxygen concentration [30]. Therefore, this non-invasive technique has been used to estimate free radical reactions [31–33], the redox state [34,35], and antioxidant activity [36] in various animal models including ischemia-reperfusion [37], hypoxia-hyperoxia [32,37,38], x-ray irradiation [39] and streptozotocin-induced diabetes [40]. In this study, we used in vivo ESR measurement to evaluate in vivo free radical reactions catalyzed by iron, in order to develop a non-invasive technique for studying iron-induced diseases and their therapy, and to clarify the role of low molecular weight iron in oxidative stress. This is the first report on real-time, non-invasive in vivo free radical reactions caused by iron and the relationship between catalytic activity and the form of iron. MATERIALS AND METHODS
Chemicals 3-Carbamoyl-2,2,5,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and Trolox C, a water soluble vitamin E analogue, were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). The carbamoylPROXYL was dissolved in deionized water (Milli Q) to 300 mM. The Trolox was dissolved in 5% sodium bicarbonate and adjusted to pH 7.0 with 1 M HCl just before use. Ferric citrate was obtained from Sigma Chemical Co. (St. Louis, MO, USA). It was dissolved with 0.1 M HCl and adjusted to pH 7.0 with NaOH. Desferrioxamine (Desferal) was obtained from CibaGeigy (Japan). It was freshly prepared by dissolving it in normal saline to 100 mM. All the other chemicals were obtained from either Sigma Chemical Co. (St. Louis, MO, USA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Iron overload and in vivo ESR measurement Female ddy mice (age 3 weeks, 10 –14 g) were obtained from Seac Yoshitomi Co. (Fukuoka, Japan), and were acclimatized for 1 week before the experiments. Diet (MF, Oriental Yeast Co. Tokyo, Japan) and water were provided ad lib. After anesthesia by an intramuscular injection of pentobarbital (50 mg/g body weight [b.wt.], Nembutal, Abbott Laboratories, North Chicago,
IL, USA), the mice were subcutaneously loaded with ferric-citrate (0.2 mmol/g b.wt.). Carbamoyl-PROXYL (0.75 mmol/g b.wt.) was injected into the tail vein of mice 10, 30, and 60 min after iron loading. Immediately after injection of the nitroxyl probe, ESR spectra were measured in the upper abdomen of the mice with a JEOL, JES-PE-1X ESR spectrometer equipped with an L-band microwave power unit (ES-LBIC) and a loop-gap resonator (diameter 33 mm and length 5 mm; JEOL, Japan). The microwave frequency was about 1.1 GHz and the power was 1.0 mW. The amplitude of the 100 kHz field modulation was 0.125 mT. The external magnetic field was swept at a scan rate of 5 mT/min. The initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection. The effects of antioxidants on signal decay were investigated by administering Trolox (0.4 mmol/g b.wt.) IP or 50% DMSO (37 mmol/g b.wt.) 10 min before iron loading. Effect of desferrioxamine (DF) on signal decay, lipid peroxidation, and tissue iron content A single dose of DF (0.19 mmol/g b.wt.) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. The signal decay was measured 10, 30, and 60 min after iron loading in each group. Ninety min after iron loading, the mice were sacrificed by exsanguination from the abdominal aorta, that was then perfused with 4 –5 ml of normal saline before removing the liver, kidney, and spleen to measure lipid peroxidation and iron content. Urine was collected from the urinary bladder and diluted with 50 mM HCl. Lipid peroxidation The concentration of thiobarbituric reactive substances (TBARs) in the liver was measured spectrophotometrically using a modification of the methods of Asakawa et al. [41] and Uchiyama et al. [42]. The liver was homogenized (1:10, W/V) in 1.15% KCl and 5 mM BHT. A 0.5 ml homogenate was mixed to give a final concentration of 2.2% TCA, 0.5 mM EDTA, and 0.8% SDS, and then reacted with 0.2% thiobarbituric acid (TBA) in a boiling water bath for 45 min. After cooling, the chromogen was extracted in n-butanol. The concentration of TBARs was calculated from the difference between the absorption values of the butanol-extracted supernatant at 532 and 520 nm. 1,1,3,3,-Tetraethoxypropane was used as the standard. Iron content The amounts of non-heme iron in the liver, kidney, and spleen were determined using a modification [43] of
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the method of Foy et al. [44]. Briefly, tissue samples were homogenized (1:5, W/V) with 0.15 M NaCl in 10 mM NaOH-Hepes buffer (pH 7.0). Non-heme iron was extracted 3 times by boiling 0.5 ml samples of homogenate with 1 ml acid mixture (12.5% TCA and 2% sodium pyrophosphate) for 15 min. One ml of acid extract or diluted urine was mixed with 2 ml of color reagent (1 mM ferrozine in 50 mM sodium ascorbate and 1.05 M sodium acetate) and left at room temperature for 15 min before reading the absorbance at 562 nm. In vitro antioxidant properties of carbamoyl-PROXYL The in vitro experiment was carried out using liver homogenate (1:10, W/V of 1.15% KCl). Half ml of liver homogenate was incubated with 50 mM ferric citrate or antioxidants at 37°C for 1 h, and the reaction was stopped with 5 mM BHT. Lipid peroxidation was determined. Statistical analysis Statistical analyses were carried out using Stat View-J 4.02. The data were analyzed by a one way analysis of variance (ANOVA), with Dunn’s test used as a post test. The 0.05 level was selected as the minimum level of statistical significance. All the results are presented as the mean 6 standard error (SE). RESULTS
Figure 1A shows a typical ESR spectrum of carbamoyl-PROXYL observed in the upper abdomen of a mouse. The isotropic triplet sharp lines indicate that the nitroxyl probe exists as a free monomer. The three lines were not symmetric and showed a little signal distortion, that is often observed in in vivo ESR measurement. The signal intensity gradually decreased, obeying first order kinetics as described previously [33]. The intact and reduced forms of carbamoyl-PROXYL were mostly recovered from liver and kidney after intravenous injection into mice [45], indicating that the signal of nitroxyl probe shown in Fig. 1A arises mainly from the liver. The decay curves of carbamoyl-PROXYL in control and ironloaded mice are shown in Fig. 1B. The decay constants were calculated from the initial slope of the curves. Table 1 gives the signal decay constants of carbamoylPROXYL in the control, ferric citrate, and antioxidanttreated mice. Treatment with a high dose (0.2 mmol/g) of iron significantly enhanced the signal decay, whereas the lower dose (0.05 mmol/g) did not. Citrate itself did not affect signal decay. The enhanced signal decay caused by iron loading was significantly inhibited by pre-treatment with the chain-breaking antioxidant Trolox (0.4 mmol/g),
Fig. 1. ESR spectrum of carbamoyl-PROXYL in the upper abdomen of mouse (A) and signal decay curve in control and iron overloaded mice (B). Mice were subcutaneously loaded with ferric-citrate (0.2 mmol/g body wt) or saline. Carbamoyl-PROXYL (0.75 mmol/g body wt) was injected into the tail vein of mice 10 min after iron loading. Immediately after injection of the nitroxyl probe, ESR spectra were measured in the upper abdomen of the mice with an L-band ESR spectrometer. The upper in (A) demonstrates the structure of carbamoyl-PROXYL.
although Trolox itself had no direct effect on the signal decay. Only the high dose of DMSO (37 mmol/g) showed a slight inhibitory effect on iron. The relationship of iron to the signal decay was investigated by examining the effect of an iron chelator, desferrioxamine (DF). Desferrioxamine was given to iron-overloaded mice at various times relative to the iron loading, and the signal decay was determined 10, 30, and 60 min after iron loading in each group (Table 2). The signal decay
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Table 1. Effect of Iron Overloading on Signal Decay Constant of Carbamoyl-PROXYL in Upper Abdomen of Mice Signal Decay Constant (min21) Control (nontreatment) Control (citrate 0.2 mmol/g) Fe-citrate 0.05 mmol/g Fe-citrate 0.2 mmol/g 1 Trolox (0.4 mmol/g) 1 DMSO (37 mmol/g) Trolox (0.4 mmol/g)
0.174 6 0.006 (11)a,b,c 0.179 6 0.004 (3) 0.176 6 0.008 (3) 0.275 6 0.010 (12)a,d 0.218 6 0.012 (12)b,d 0.230 6 0.012 (6)c 0.180 6 0.009 (4)
Mice were subcutaneously loaded with ferric citrate or citrate, and Trolox or DMSO in Fe-citrate treated group were intraperitoneally injected 10 min before iron loading. Carbamoyl-PROXYL (0.75 mmol/g) was intravenously administered 10 min after Fe-citrate or citrate loading, or 20 min after treatment with only Trolox. ESR spectrum was immediately observed in upper abdomen of mice, and the signal decay constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection, as described in the legend of Fig. 1. Results are presented as mean 6 SE (n) Significant differences are indicated by different letter at ap , 0.0001, dp , .010 and b,cp , .05.
constants after DF treatment in iron-overloaded mice are shown in the dotted box. The effects of DF strongly depended on the time it was administered to the iron-overloaded group. Iron overloading alone increased the signal decay to the same extent at 10 and 30 min, and slightly decreased it to 0.239 at 60 min (p , .05), indicating the lasting effect of iron on signal decay. The pre- and simultaneous treatment of DF decreased the signal decay significantly (p , .0001) to the control level at all times, and DF suppressed the effect of iron by 92% and 100% in these treatments, respectively. The effect of DF was still observed 60 min after the treat-
ment, indicating that the effect is persistent. DF itself had no effect on the signal decay in control mice. When DF was given 20 min after iron loading, the signal decay was also significantly decreased (p , .0001) compared with the value before treatment, and DF suppressed the effect of iron by about 86%. When DF was given 50 min after iron loading, the signal decay decreased significantly (p , .05) from the pretreatment level and the suppressive effect of DF was 59%. Although the effect of DF apparently decreased in the treatment at 50 min, significant amounts of iron responsible for the signal decay were present as a DF chelatable form. The relationship between chelatable iron and the signal decay was also confirmed by measuring the liver iron content and iron excretion by the kidney into the urine. The liver iron content was significantly decreased in DF-treated groups compared with the untreated group (Fig. 2A). The greatest effect of DF was observed in the simultaneous treatment and the 20 min after iron loading groups (p , .0001). The DF treatment 50 min after iron loading showed a lower but still significant effect (p , .05). Figure 2B shows a good correlation between the liver iron content and the signal decay constant 60 min after iron loading (r 5 0.817, p , .001). Desferrioxamine-enhanced iron excretion into the urine significantly (Fig. 3A). The simultaneous and 20 min treatments showed the greatest effect (p , .0001). The effect was lower, but still significant, with the 50 min treatment (p , .01), suggesting that the amount of iron in the chelatable pool decreased gradually. There was no significant difference in sum of iron in the liver, kidney, and urine among iron-overloaded mice (2.21 6 0.18 mmol) and iron
Table 2. Effect of Desferrioxamine (DF) Treatment on Signal Decay Constant of Carbamoyl-PROXYL in Upper Abdomen of Mice Signal Decay Constant (min21) Time After Iron loading 10 min Control Iron overloaded
Simultaneous treatment
0.268 6 0.014
0.188 6 0.020
0.165 6 0.016
0.161 6 0.011
0.213 6 0.009 ............................................. 0.238 6 0.016e d
.....
50 min after iron loading
0.182 6 0.020
0.175 6 0.011b .................................................. 0.283 6 0.0016d
........
20 min after iron loading
0.184 6 0.014a
0.210 6 0.009 0.194 6 0.009e
.............................
Pre-treatment
60 min
0.176 6 0.01 0.170 6 0.014 0.163 6 0.021 0.192 6 0.002 0.181 6 0.006 0.162 6 0.002 0.281 6 0.009a,b 0.266 6 0.006c 0.239 6 0.011c .......................................................................................................................................
.................
No DF treatment DF treatment No DF treatment DF treatment
30 min
Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) at time 0 min. Control mice were injected with saline only. A single dose of DF (0.19 mmol/g) was intraperitoneally administrated 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. Signal decay constant of carbamoyl-PROXYL in upper abdomen was determined 10, 30, 60 min after iron loading, as described in Table 1. Results are presented as mean 6 SE of 4 –7 mice. The dotted box emphasizes that these decay constants are obtained after DF treatment in iron overloaded mice. Significant differences are indicated by different letters at a,b,dp , .0001 and c,ep , .05.
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Fig. 2. Effect of desferrioxamine (DF) on liver iron content in iron overloaded mice (A) and the correlation between liver iron content and signal decay constant of carbamoyl-PROXYL (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) and a single dose of DF (0.19 mmol/g) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. ESR signal of carbamoyl-PROXYL was observed 10, 30, and 60 min after iron loading, and the initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection, as described in the legend of Fig. 1. Liver iron content was determine 90 min after iron loading. Results are presented as mean 6 SE of 4 –7 mice. Significant differences are: *p , .05; **p , .01; ***p , .0001 compared with control; and #p , .05 compared with DF simultaneous treatment. The correlation line was obtained by regression analysis (r 5 0.817, p , .001).
overloaded mice treated with DF (1.96 6 0.25 mmol for pre-, 2.21 6 0.19 mmol for simultaneous, 2.11 6 0.15 mmol for 20 min after and 2.06 6 0.19 mmol for 50 min after iron loading), suggesting that urinary excretion is the major pathway for eliminating the iron-DF complexes, although iron should be eliminated through biliary excretion in the absence of DF [25]. The plot of the signal decay suppressed by DF and the amount of iron excretion in each mouse showed quite a good correlation (r 5 0.827, p , .001, Fig. 3B). The results clearly demonstrated that the enhanced signal decay was caused by “chelatable iron.” Iron toxicity was evaluated by measuring liver TBARs. The liver TBARs were significantly increased in the iron-overloaded mice and the levels were correlated with the liver iron content (r 5 0.734, p , .001). Lipid peroxidation was inhibited by treatment with DF either before or 50 min after iron loading (Fig. 4A). The inhibitory effect was higher with the pre-treatment, indicating that lipid peroxidation was rapidly initiated by chelatable iron. To confirm the relationship between free radical reactions and the enhanced signal decay with in vivo ESR measurement, the effect of the nitroxyl probe on lipid peroxidation was determined. If the reaction of the nitroxyl probe producing the ESR signal decay is involved in free-radical-mediated lipid peroxidation, then the nitroxyl probe should suppress lipid peroxidation. The
iron-promoted lipid peroxidation was inhibited by the nitroxyl probe (Fig. 4). The probe significantly suppressed liver TBARs in the iron-overloaded mice (109.6 6 6.0 and 87.8 6 3.7 nmol/g liver without and with nitroxyl probe, respectively, p , .001), although there was no significant difference in the liver iron content with or without the nitroxyl probe (data not shown). The effect of the nitroxyl probe on lipid peroxidation in vivo seemed to be additive with DF but not with Trolox. These facts strongly indicate that the nitroxyl probe must be involved in the free radical reactions responsible for lipid peroxidation caused by iron loading. The antioxidant activity of the nitroxyl probe was also confirmed with an in vitro experiment using liver homogenate (Table 3). Lipid peroxidation in the homogenate was induced by incubating it with 50 mM Fe(III)citrate and TBARs were suppressed by either DF (30 mM), carbamoyl-PROXYL (150 mM) or Trolox (150 mM). Co-incubation of DF or Trolox with the nitroxyl probe inhibited the TBARs relative to the control level, indicating the antioxidant effect of the nitroxyl probe. DISCUSSION
The in vivo signal decay of nitroxyl probe is reported to be related to physiological reducing capacity and free radical reactions in the body [31–39]. Intravenous injection of
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Fig. 3. Effect of desferrioxamine (DF) on iron excretion in iron overloaded mice (A) and the correlation between excreted iron content and suppression of enhanced signal decay by DF (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) and a single dose of DF (0.19 mmol/g) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. ESR signal of carbamoyl-PROXYL was observed 10, 30, and 60 min after iron loading, and the initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity versus time after injection, as described in the legend of Fig. 1. The iron contents in kidney (u) and urine (h) were determined 90 min after iron loading. Results are presented as mean 6 SE of 4 –7 mice. Significant differences are *p , .05, **p , .01, ***p , .0001, compared with control and ##p , .05 compared with DF simultaneous treatment. The data used for correlation analysis were obtained from the difference of signal decay constant between before and after DF treatment, and excreted iron contents were measured with the same individual mice used for ESR experiment. The line was obtained by regression analysis (r 5 0.827, p , .001).
Fig. 4. In vivo effect of carbamoyl-PROXYL on lipid peroxidation of mice liver in the presence of DF (A) and Trolox (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g). Mice in the group of nitroxyl probe treatment (u) were intravenously injected with three doses of carbamoyl-PROXYL (0.75 mmol/g/dose) 10, 30, and 60 min after iron loading and then liver TBARs were estimated with its homogenate 90 min after iron loading as described in Methods. A single dose of DF (0.19 mmol/g) (A) or Trolox (0.4 mmol/g) (B) were given to iron overloaded mice 10 min before or 50 min after iron loading. Mice in the group of nitroxyl untreatment (■) were done as the same procedure as the nitroxyl treatment group without injection of the nitroxyl probe. Results are presented as mean 6 SE of 5–12 mice. Significant difference are *p , .05 and **p , .01 and between the groups indicated.
In vivo detection of Fe toxicity Table 3. In Vitro Antioxidant Activity of Carbamoyl-PROXYL on Lipid Preoxidation TBARs (nmol/ml) (a) Control Fe-citrate 50 mM 1 DF 30 mM 1 carbamoyl-PROXYL 150 mM 1 DF 30 mM and carbamoyl-PROXYL 150 mm (b) Control Fe-citrate 50 mM 1 Trolox 150 mM 1 carbamoyl-PROXYL 150 mM 1 Trolox 150 mM and carbamoyl-PROXYL 150 mM
0.74 6 0.35 17.50 6 0.007 6.77 6 0.001 9.41 6 0.027 0.50 6 0.014 0.68 6 0.033 12.60 6 0.001 2.66 6 0.000 4.10 6 0.001 0.76 6 0.006
Lipid peroxidation of liver homogenate was induced by incubation with 50 mM ferric citrate at 37°C for 1 h in the presence or absence of DF (30 mM) (a), Trolox (150 mM) (b), and carbamoyl-PROXYL (150 mM). TBARs were determined with the liver homogenate as described in Materials and Methods. Results are presented as mean 6 SE of duplicate.
nitroxyl probe in mice or rats produced triplet lines in the in vivo ESR measurement and the signal decay obeyed first order kinetics. The signal decay was increased by oxidative stress such as hyperoxia [32], ischemia-reperfusion [37], and x-irradiation [39], in disease models such as liver damage induced by CCl4 [32] and streptozotocin-induced diabetes [40]. In addition, the enhancement was suppressed by in vivo antioxidants [31,32,36,39]. In vitro experiments have also shown that nitroxyl probe reacts with various free radical species such as superoxide, hydroxyl, and peroxyl radicals [46]. The chain-breaking antioxidant property of water-soluble nitroxyl probe has been observed in liver microsome with NADPH [47,48]. This evidence strongly suggests that nitroxyl probe is very susceptible to free radical reactions involved in oxidative damage. The single dose of excess ferric citrate rapidly increased the in vivo signal decay, that was suppressed by both iron chelator and the chain-breaking antioxidant, suggesting a relationship between signal decay and free radical reactions catalyzed by iron. Indeed, the nitroxyl probe showed in vivo antioxidant activity by inhibiting lipid peroxidation (TBARs) in iron-overloaded mice, and this was confirmed by an in vitro experiment. Therefore, the enhanced signal decay produced by iron should be related to free-radical-mediated lipid peroxidation. It is widely believed that iron causes complications in several diseases by catalyzing free radical reactions that produce cell and tissue injuries [1]. Iron toxicity is not only involved in hemochromatosis, but also in cancer, ischemia, Parkinson’s Disease, and rheumatoid arthritis. Iron exists in various forms in biological systems, including heme iron, iron bound to proteins such as ferritin, hemosiderin, and transferrin, and “low molecular weight iron” such as iron
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ions attached to ATP, citrate, deoxyribose, and membrane lipids. The most interesting form of iron is the “low molecular weight pool,” because it is a catalytic iron responsible for toxic oxygen generation. Low molecular weight iron has been found in both the extracellular and intracellular compartments in iron-overloaded patients and animals [15–18], and also in many pathological conditions such as alcoholism, and ischemia-reperfusion [19–22]. In high iron overload, the capability of transferrin to maintain iron is exceeded, resulting in excessive amounts of low molecular weight iron in the blood compartment. Hepatocytes and macrophages rapidly take up the excess iron, resulting in expansion of the transit intracellular low molecular weight iron pool. Chronic and acute iron-overloaded animals have been used as models to investigate the mechanism of iron toxicity. Most of the animal experiments have evaluated the presumed effect of iron via free radical reactions by following changes in antioxidant enzyme activity, antioxidant status, and lipid peroxidation level [5–8]. However, there is little direct evidence that proves iron catalyzes free radical reactions and identifies the catalytic iron in vivo. The formation of iron-dependent oxygen free radicals by low molecular weight iron in biological systems is still controversial. Although “low molecular weight iron” has been identified as “Fenton-catalytic” iron, and the “bleomycin assay” has been developed to measure the availability of catalytic iron in biological samples [49], there is no direct proof that bleomycin-detectable iron and low molecular weight iron catalyze free radical reactions in vivo. Moreover, although the Fenton reaction is thought to produce hydroxyl radicals as the reactive species, it is also suggested that other species are formed, such as ferryl radical [12,14]. In addition, the reaction of iron complex with lipid hydroperoxides is an order of magnitude faster than the reaction with hydrogen peroxide [50,51], suggesting that, in biological systems, the direct reaction with lipid hydroperoxide is more likely to occur than that through the Fenton reaction. The only direct measurements involve in vivo spin trapping of methyl radicals, produced secondary from hydroxyl radicals with DMSO, and in vitro detection with X-band ESR spectroscopy [23–25]. This technique clearly demonstrated the in vivo generation of hydroxyl radical in acute and dietary iron-overloaded rats. Although the spintrapping technique suggest the significance of Fenton reaction in iron overloaded animals, the other reactions catalyzed by iron in vivo should be also considered. In addition, the association between catalytic activity and the form of iron in vivo is not well established. Therefore, direct and real-time determination of in vivo free radical reactions caused by iron is essential to better understanding the mechanism of its toxicity. The in vivo ESR measurement with a nitroxyl probe is a very suitable technique to investigate the mechanism of iron toxicity in vivo.
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Iron kinetic data suggest that low molecular weight iron is transiently present after a single injection of iron [52]. Desferrioxamine is an effective iron chelator. It readily chelates iron from the low molecular weight iron pool, and its complexes are rapidly excreted into the bile or urine, depending on the source of the iron [53,54]. Urinary iron appears to be derived from the “low molecular weight iron” in the plasma or the transit exchangeable pool in the liver, whereas biliary iron arises from ferritin degradation in the liver. Therefore, the experiments with DF should be able to estimate the time-dependence of the amount of low molecular weight iron. Because the sum amount of iron in the liver, kidney, and urine of iron-overloaded mice was no different in the DF-treated and non-treated groups, most of the iron-DF complexes were probably excreted into the urine. Although all of the DF treatments had an effect on signal decay, lipid peroxidation, and iron status, the effect was lower in the treatment 50 min after iron loading, suggesting the relatively short life of “chelatable iron” or “low molecular weight iron.” The signal decay suppressed by DF was very well correlated with the amount of urinary iron in iron overloaded mice, indicating that “chelatable iron” contributes to the enhanced signal decay. There was also a good correlation between the in vivo signal decay, iron content, and lipid peroxidation in the liver. These in vivo findings support the hypothesis that “low molecular weight iron” catalyzes free-radicalmediated lipid peroxidation. The enhanced signal decay caused by iron was suppressed by the chain-breaking antioxidant Trolox, and to a lesser degree by the hydroxyl radical scavenger DMSO. Iron preferentially stimulates peroxidation by lipid decomposition though reactions (1) and (2) [50], because there is usually plenty of hydroperoxide present in the cell membranes and plasma lipid component. Garnier-Suillerot et al. [51] reported that the kinetic constant of reaction (I) is 1.5 3 103 M21s21, that is higher than that of the Fenton reaction (76 M21s21). The reduced effect of DMSO, a hydroxyl radical scavenger, implies a smaller contribution of hydroxyl radical to the in vivo oxidative stress in the liver caused by iron. iron complex
2LOOH O ¡ LO 1 LO 2 1 H 2 O
(1)
LOOH 1 Fe 21 3 LO • 1 OH 2 1 Fe 31
(2)
Our results strongly indicate that in vivo ESR spectroscopy using a nitroxyl probe provides information about the catalytic form and activity of iron. They support the hypothesis that chelatable or low molecular weight iron plays a role in free radical reactions and lipid peroxidation. Iron should initiate lipid peroxidation pref-
erentially via reaction with lipid hydroperoxide and produce peroxyl and alkoxyl radicals. This hypothesis is supported by the effect of the chain-breaking antioxidant. The technique of in vivo ESR spectroscopy discussed here may be applied to evaluate new iron chelators and antioxidants for the therapy of iron-related diseases, such as hemochromatosis. Acknowledgements — This work was supported by a Grant-in-Aid for Research on Priority Areas, a Grant-in-Aid for Co-operative Research, a Grant-in-Aid for Developmental Scientific Research, a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan, by the Neito Foundation, and by the Cosmetology Research Foundation.
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[37] Utsumi, H.; Takeshita, K.; Miura, Y.; Masuda, S.; Hamada, A. In vivo ESR measurement of radical reaction in whole mice. Influence of inspired oxygen and ischemia-reperfusion injury on nitroxide reduction. Free Radic. Res. Commun. 19:219 –225; 1993. [38] Miura, Y.; Utsumi, H.; Hamada, A. Effect of inspired oxygen on in vivo redox reaction of nitroxide radicals in whole mice. Biochem. Biophys. Res. Commun. 182:1108 –1114; 1992. [39] Miura, Y.; Anzai, K.; Urano, S.; Ozawa, T. In vivo electron paramagnetic resonance studies on oxidative stress caused by x-irradiation in whole mice. Free Radic. Biol. Med. 23:533–540; 1997. [40] Sano, T.; Uneda, F.; Hashimoto, T.; Nawata, H.; Utsumi, H. Oxidative stress measurement by in vivo electron spin resonance spectroscopy in rats with sereptozotocin-induced diabetes. Diabetologia 41:1355–1360; 1998. [41] Asakawa, T.; Matsushita, S. Coloring condition of thiobarbituric acid test for detecting lipid hydroperoxide. Lipids 15:137–140; 1980. [42] Uchiyama, M.; Mihara, M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem. 86: 271–278; 1978. [43] Simpson, R. J.; Peters, T. J. Forms of soluble iron in mouse stomach and duodenal lumen: significance of mucosal uptake. Br. J. Nutr. 63:79 – 89; 1990. [44] Foy, A. L.; Williams, H. L.; Cortell, S.; Conrad, M. E. A modification procedure for determination of non-heme iron in tissue. Anal. Biochem. 18:559 –563; 1967. [45] Sano, H.; Matsumoto, K., Utsumi, H. Synthesis and imaging of blood-brain-barrier permeable nitroxyl probes for free radical reactions in brain of living mice. Biochem Mol Biol Int. 42:641– 647; 1997. [46] Kocherginsky, N.; Swartz, H. M. Chemical reactivity of nitroxides. In: Swartz, H. M., eds. Nitroxide spin labels: reaction in biology and chemistry. Boca Raton, FL: CRC Press Inc.; 1995: 27– 66. [47] Miura, Y.; Utsumi, H.; Hamada, A. Antioxidant activity of nitroxide radical in lipid peroxidation of rat liver microsomes. Arch. Biochem. Biophys. 300:148 –156; 1993. [48] Nilsson, U. F.; Olsson, L.; Carlin, G.; Bylund-Fellenius, A. C. Inhibition of lipid peroxidation by spin labels: relationship between structure and function. J. Biol. Chem. 264:11131–11135; 1989. [49] Gutteridge, J. M. C.; Rowly, D. A; Halliwell, B. Superoxidedependent formation of hydroxyl radicals in the presence of iron salts. Detection of “free” iron in biological systems by using bleomycin-dependent degradation of DNA. Biochem J. 199:263– 265; 1981. [50] Gutteridge, J. M. C.; Halliwell, B. Iron toxicity and oxygen radical. Baillieres Clin. Haematol. 2:195–256; 1989. [51] Garnier-Suillerot, A.; Tosi, L.; Paniago, E. Kinetic and mechanism of vesical lipoperoxide decomposition by Fe(II). Biochim. Biophys. Acta. 794:307–312; 1984. [52] Bothwell, T. H.; Charlton, R. W.; Cook, J. D.; Finch, C. A. Iron metabolism in man. 1st ed. Oxford: Blackwell Scientific Publication; 1979. [53] Hershko, C. Biological model for studying iron chelating drugs. Baillieres Clin. Haematol. 2:293–321; 1989. [55] Pippard, M. J. Desffioxamine-induced iron excretion in humans. Baillieres Clin. Haematol. 2:323–343; 1989.
ABBREVIATIONS
BHT— butyl hydroxy toluene Carbamoyl-PROXYL—3-Carbamoyl-2,2,5,5,5-tetramethylpyrrolidine-1-oxyl DF— desferrioxamine DMSO— dimethyl sulfoxide TBARs—thiobarbituric acid reactive substances
PII S0891-5849(98)00314-1
Original Contribution NONINVASIVE EVALUATION OF IN VIVO FREE RADICAL REACTIONS CATALYZED BY IRON USING IN VIVO ESR SPECTROSCOPY NOPPAWAN PHUMALA, TATSUO IDE,
and
HIDEO UTSUMI
Department of Biophysics, Faculty of Pharmaceutical Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan (Received 10 August 1998; Revised 26 October 1998; Accepted 26 October 1998)
Abstract—The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 mmol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that “chelatable iron” contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron. © 1999 Elsevier Science Inc. Keywords—Iron overload, In vivo ESR, Nitroxyl probe, Desferrioxamine, Free radicals
INTRODUCTION
actions [5– 8]. Because low molecular weight forms of iron have been detected in iron overload [9 –12] and other oxidative stress related conditions [13–16], this form of iron may play a catalytic role in free radical reactions. Although the spin trapping technique using X-band ESR provides information about species of free radicals in vitro [1,17,18], the formation of hydroxyl radicals by iron is controversial [1,19 –22]. Unfortunately, there are few reports that provide direct evidence of iron-induced free radical reactions in vivo. Recently, the technique of secondary radical spin trapping has been introduced to study free radical reactions in animal models. The detection of the DMSO-derived PBN/ • CH3 radical adduct in bile samples has provided indirect evidence of hydroxyl radical generation in iron-overloaded rats [23–25]. However, this technique is limited to reactions that presumably occur in the liver and excrete the spin adduct into the bile. A real time and non-invasive measurement of in vivo free radical reactions in iron overloaded animals is quite important to fulfill the information about association between catalytic activity and toxic form of iron in vivo, although spin-trapping technique clearly clarifies reactive oxygen species.
Iron is an essential element for biological functions in aerobic organisms, but there is growing evidence that implicates iron in several diseases, including cancer and arthritis [1,2]. Hemochromatosis is a classic example of iron toxicity. The massive iron overload in the vital organs, including the endocrine glands, heart, and liver, causes organ dysfunction and hepatic cirrhosis [3]. The major causes of death in inherited hemochromatosis patients are cardiac disease, liver failure, and hepatocellular carcinoma [4]. It has long been suspected that free radicals play a role in iron toxicity, because of their powerful oxidant activity, that has been demonstrated in in vitro experiments. Iron-induced lipid peroxidation and cellular injuries were prevented by antioxidants, presumably involving free radical reactions. Changes in the level of antioxidants and antioxidant enzymes imply ongoing free radical reAddress correspondence to: Hideo Utsumi, Department of Biophysics, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Tel: 181 92-642-6621; Fax: 181 92-642-6626; E-Mail: [email protected]. 1209
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In vivo ESR spectroscopy (L-band and 300 MHZ) has recently been developed to measure free radicals in living animals. Because its sensitivity relative to the amounts of endogenous free radical produced in vivo is poor, nitroxyl stable radicals (piperidine-N-oxyl and pyrrolidine-N-oxyl derivatives) have been used as spin probes for in vivo ESR measurement. The decay of nitroxyl ESR signals is influenced by the generation of active oxygen species [26], the biological redox system [27–29] and oxygen concentration [30]. Therefore, this non-invasive technique has been used to estimate free radical reactions [31–33], the redox state [34,35], and antioxidant activity [36] in various animal models including ischemia-reperfusion [37], hypoxia-hyperoxia [32,37,38], x-ray irradiation [39] and streptozotocin-induced diabetes [40]. In this study, we used in vivo ESR measurement to evaluate in vivo free radical reactions catalyzed by iron, in order to develop a non-invasive technique for studying iron-induced diseases and their therapy, and to clarify the role of low molecular weight iron in oxidative stress. This is the first report on real-time, non-invasive in vivo free radical reactions caused by iron and the relationship between catalytic activity and the form of iron. MATERIALS AND METHODS
Chemicals 3-Carbamoyl-2,2,5,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and Trolox C, a water soluble vitamin E analogue, were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). The carbamoylPROXYL was dissolved in deionized water (Milli Q) to 300 mM. The Trolox was dissolved in 5% sodium bicarbonate and adjusted to pH 7.0 with 1 M HCl just before use. Ferric citrate was obtained from Sigma Chemical Co. (St. Louis, MO, USA). It was dissolved with 0.1 M HCl and adjusted to pH 7.0 with NaOH. Desferrioxamine (Desferal) was obtained from CibaGeigy (Japan). It was freshly prepared by dissolving it in normal saline to 100 mM. All the other chemicals were obtained from either Sigma Chemical Co. (St. Louis, MO, USA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Iron overload and in vivo ESR measurement Female ddy mice (age 3 weeks, 10 –14 g) were obtained from Seac Yoshitomi Co. (Fukuoka, Japan), and were acclimatized for 1 week before the experiments. Diet (MF, Oriental Yeast Co. Tokyo, Japan) and water were provided ad lib. After anesthesia by an intramuscular injection of pentobarbital (50 mg/g body weight [b.wt.], Nembutal, Abbott Laboratories, North Chicago,
IL, USA), the mice were subcutaneously loaded with ferric-citrate (0.2 mmol/g b.wt.). Carbamoyl-PROXYL (0.75 mmol/g b.wt.) was injected into the tail vein of mice 10, 30, and 60 min after iron loading. Immediately after injection of the nitroxyl probe, ESR spectra were measured in the upper abdomen of the mice with a JEOL, JES-PE-1X ESR spectrometer equipped with an L-band microwave power unit (ES-LBIC) and a loop-gap resonator (diameter 33 mm and length 5 mm; JEOL, Japan). The microwave frequency was about 1.1 GHz and the power was 1.0 mW. The amplitude of the 100 kHz field modulation was 0.125 mT. The external magnetic field was swept at a scan rate of 5 mT/min. The initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection. The effects of antioxidants on signal decay were investigated by administering Trolox (0.4 mmol/g b.wt.) IP or 50% DMSO (37 mmol/g b.wt.) 10 min before iron loading. Effect of desferrioxamine (DF) on signal decay, lipid peroxidation, and tissue iron content A single dose of DF (0.19 mmol/g b.wt.) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. The signal decay was measured 10, 30, and 60 min after iron loading in each group. Ninety min after iron loading, the mice were sacrificed by exsanguination from the abdominal aorta, that was then perfused with 4 –5 ml of normal saline before removing the liver, kidney, and spleen to measure lipid peroxidation and iron content. Urine was collected from the urinary bladder and diluted with 50 mM HCl. Lipid peroxidation The concentration of thiobarbituric reactive substances (TBARs) in the liver was measured spectrophotometrically using a modification of the methods of Asakawa et al. [41] and Uchiyama et al. [42]. The liver was homogenized (1:10, W/V) in 1.15% KCl and 5 mM BHT. A 0.5 ml homogenate was mixed to give a final concentration of 2.2% TCA, 0.5 mM EDTA, and 0.8% SDS, and then reacted with 0.2% thiobarbituric acid (TBA) in a boiling water bath for 45 min. After cooling, the chromogen was extracted in n-butanol. The concentration of TBARs was calculated from the difference between the absorption values of the butanol-extracted supernatant at 532 and 520 nm. 1,1,3,3,-Tetraethoxypropane was used as the standard. Iron content The amounts of non-heme iron in the liver, kidney, and spleen were determined using a modification [43] of
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the method of Foy et al. [44]. Briefly, tissue samples were homogenized (1:5, W/V) with 0.15 M NaCl in 10 mM NaOH-Hepes buffer (pH 7.0). Non-heme iron was extracted 3 times by boiling 0.5 ml samples of homogenate with 1 ml acid mixture (12.5% TCA and 2% sodium pyrophosphate) for 15 min. One ml of acid extract or diluted urine was mixed with 2 ml of color reagent (1 mM ferrozine in 50 mM sodium ascorbate and 1.05 M sodium acetate) and left at room temperature for 15 min before reading the absorbance at 562 nm. In vitro antioxidant properties of carbamoyl-PROXYL The in vitro experiment was carried out using liver homogenate (1:10, W/V of 1.15% KCl). Half ml of liver homogenate was incubated with 50 mM ferric citrate or antioxidants at 37°C for 1 h, and the reaction was stopped with 5 mM BHT. Lipid peroxidation was determined. Statistical analysis Statistical analyses were carried out using Stat View-J 4.02. The data were analyzed by a one way analysis of variance (ANOVA), with Dunn’s test used as a post test. The 0.05 level was selected as the minimum level of statistical significance. All the results are presented as the mean 6 standard error (SE). RESULTS
Figure 1A shows a typical ESR spectrum of carbamoyl-PROXYL observed in the upper abdomen of a mouse. The isotropic triplet sharp lines indicate that the nitroxyl probe exists as a free monomer. The three lines were not symmetric and showed a little signal distortion, that is often observed in in vivo ESR measurement. The signal intensity gradually decreased, obeying first order kinetics as described previously [33]. The intact and reduced forms of carbamoyl-PROXYL were mostly recovered from liver and kidney after intravenous injection into mice [45], indicating that the signal of nitroxyl probe shown in Fig. 1A arises mainly from the liver. The decay curves of carbamoyl-PROXYL in control and ironloaded mice are shown in Fig. 1B. The decay constants were calculated from the initial slope of the curves. Table 1 gives the signal decay constants of carbamoylPROXYL in the control, ferric citrate, and antioxidanttreated mice. Treatment with a high dose (0.2 mmol/g) of iron significantly enhanced the signal decay, whereas the lower dose (0.05 mmol/g) did not. Citrate itself did not affect signal decay. The enhanced signal decay caused by iron loading was significantly inhibited by pre-treatment with the chain-breaking antioxidant Trolox (0.4 mmol/g),
Fig. 1. ESR spectrum of carbamoyl-PROXYL in the upper abdomen of mouse (A) and signal decay curve in control and iron overloaded mice (B). Mice were subcutaneously loaded with ferric-citrate (0.2 mmol/g body wt) or saline. Carbamoyl-PROXYL (0.75 mmol/g body wt) was injected into the tail vein of mice 10 min after iron loading. Immediately after injection of the nitroxyl probe, ESR spectra were measured in the upper abdomen of the mice with an L-band ESR spectrometer. The upper in (A) demonstrates the structure of carbamoyl-PROXYL.
although Trolox itself had no direct effect on the signal decay. Only the high dose of DMSO (37 mmol/g) showed a slight inhibitory effect on iron. The relationship of iron to the signal decay was investigated by examining the effect of an iron chelator, desferrioxamine (DF). Desferrioxamine was given to iron-overloaded mice at various times relative to the iron loading, and the signal decay was determined 10, 30, and 60 min after iron loading in each group (Table 2). The signal decay
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Table 1. Effect of Iron Overloading on Signal Decay Constant of Carbamoyl-PROXYL in Upper Abdomen of Mice Signal Decay Constant (min21) Control (nontreatment) Control (citrate 0.2 mmol/g) Fe-citrate 0.05 mmol/g Fe-citrate 0.2 mmol/g 1 Trolox (0.4 mmol/g) 1 DMSO (37 mmol/g) Trolox (0.4 mmol/g)
0.174 6 0.006 (11)a,b,c 0.179 6 0.004 (3) 0.176 6 0.008 (3) 0.275 6 0.010 (12)a,d 0.218 6 0.012 (12)b,d 0.230 6 0.012 (6)c 0.180 6 0.009 (4)
Mice were subcutaneously loaded with ferric citrate or citrate, and Trolox or DMSO in Fe-citrate treated group were intraperitoneally injected 10 min before iron loading. Carbamoyl-PROXYL (0.75 mmol/g) was intravenously administered 10 min after Fe-citrate or citrate loading, or 20 min after treatment with only Trolox. ESR spectrum was immediately observed in upper abdomen of mice, and the signal decay constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection, as described in the legend of Fig. 1. Results are presented as mean 6 SE (n) Significant differences are indicated by different letter at ap , 0.0001, dp , .010 and b,cp , .05.
constants after DF treatment in iron-overloaded mice are shown in the dotted box. The effects of DF strongly depended on the time it was administered to the iron-overloaded group. Iron overloading alone increased the signal decay to the same extent at 10 and 30 min, and slightly decreased it to 0.239 at 60 min (p , .05), indicating the lasting effect of iron on signal decay. The pre- and simultaneous treatment of DF decreased the signal decay significantly (p , .0001) to the control level at all times, and DF suppressed the effect of iron by 92% and 100% in these treatments, respectively. The effect of DF was still observed 60 min after the treat-
ment, indicating that the effect is persistent. DF itself had no effect on the signal decay in control mice. When DF was given 20 min after iron loading, the signal decay was also significantly decreased (p , .0001) compared with the value before treatment, and DF suppressed the effect of iron by about 86%. When DF was given 50 min after iron loading, the signal decay decreased significantly (p , .05) from the pretreatment level and the suppressive effect of DF was 59%. Although the effect of DF apparently decreased in the treatment at 50 min, significant amounts of iron responsible for the signal decay were present as a DF chelatable form. The relationship between chelatable iron and the signal decay was also confirmed by measuring the liver iron content and iron excretion by the kidney into the urine. The liver iron content was significantly decreased in DF-treated groups compared with the untreated group (Fig. 2A). The greatest effect of DF was observed in the simultaneous treatment and the 20 min after iron loading groups (p , .0001). The DF treatment 50 min after iron loading showed a lower but still significant effect (p , .05). Figure 2B shows a good correlation between the liver iron content and the signal decay constant 60 min after iron loading (r 5 0.817, p , .001). Desferrioxamine-enhanced iron excretion into the urine significantly (Fig. 3A). The simultaneous and 20 min treatments showed the greatest effect (p , .0001). The effect was lower, but still significant, with the 50 min treatment (p , .01), suggesting that the amount of iron in the chelatable pool decreased gradually. There was no significant difference in sum of iron in the liver, kidney, and urine among iron-overloaded mice (2.21 6 0.18 mmol) and iron
Table 2. Effect of Desferrioxamine (DF) Treatment on Signal Decay Constant of Carbamoyl-PROXYL in Upper Abdomen of Mice Signal Decay Constant (min21) Time After Iron loading 10 min Control Iron overloaded
Simultaneous treatment
0.268 6 0.014
0.188 6 0.020
0.165 6 0.016
0.161 6 0.011
0.213 6 0.009 ............................................. 0.238 6 0.016e d
.....
50 min after iron loading
0.182 6 0.020
0.175 6 0.011b .................................................. 0.283 6 0.0016d
........
20 min after iron loading
0.184 6 0.014a
0.210 6 0.009 0.194 6 0.009e
.............................
Pre-treatment
60 min
0.176 6 0.01 0.170 6 0.014 0.163 6 0.021 0.192 6 0.002 0.181 6 0.006 0.162 6 0.002 0.281 6 0.009a,b 0.266 6 0.006c 0.239 6 0.011c .......................................................................................................................................
.................
No DF treatment DF treatment No DF treatment DF treatment
30 min
Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) at time 0 min. Control mice were injected with saline only. A single dose of DF (0.19 mmol/g) was intraperitoneally administrated 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. Signal decay constant of carbamoyl-PROXYL in upper abdomen was determined 10, 30, 60 min after iron loading, as described in Table 1. Results are presented as mean 6 SE of 4 –7 mice. The dotted box emphasizes that these decay constants are obtained after DF treatment in iron overloaded mice. Significant differences are indicated by different letters at a,b,dp , .0001 and c,ep , .05.
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Fig. 2. Effect of desferrioxamine (DF) on liver iron content in iron overloaded mice (A) and the correlation between liver iron content and signal decay constant of carbamoyl-PROXYL (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) and a single dose of DF (0.19 mmol/g) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. ESR signal of carbamoyl-PROXYL was observed 10, 30, and 60 min after iron loading, and the initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity vs. time after injection, as described in the legend of Fig. 1. Liver iron content was determine 90 min after iron loading. Results are presented as mean 6 SE of 4 –7 mice. Significant differences are: *p , .05; **p , .01; ***p , .0001 compared with control; and #p , .05 compared with DF simultaneous treatment. The correlation line was obtained by regression analysis (r 5 0.817, p , .001).
overloaded mice treated with DF (1.96 6 0.25 mmol for pre-, 2.21 6 0.19 mmol for simultaneous, 2.11 6 0.15 mmol for 20 min after and 2.06 6 0.19 mmol for 50 min after iron loading), suggesting that urinary excretion is the major pathway for eliminating the iron-DF complexes, although iron should be eliminated through biliary excretion in the absence of DF [25]. The plot of the signal decay suppressed by DF and the amount of iron excretion in each mouse showed quite a good correlation (r 5 0.827, p , .001, Fig. 3B). The results clearly demonstrated that the enhanced signal decay was caused by “chelatable iron.” Iron toxicity was evaluated by measuring liver TBARs. The liver TBARs were significantly increased in the iron-overloaded mice and the levels were correlated with the liver iron content (r 5 0.734, p , .001). Lipid peroxidation was inhibited by treatment with DF either before or 50 min after iron loading (Fig. 4A). The inhibitory effect was higher with the pre-treatment, indicating that lipid peroxidation was rapidly initiated by chelatable iron. To confirm the relationship between free radical reactions and the enhanced signal decay with in vivo ESR measurement, the effect of the nitroxyl probe on lipid peroxidation was determined. If the reaction of the nitroxyl probe producing the ESR signal decay is involved in free-radical-mediated lipid peroxidation, then the nitroxyl probe should suppress lipid peroxidation. The
iron-promoted lipid peroxidation was inhibited by the nitroxyl probe (Fig. 4). The probe significantly suppressed liver TBARs in the iron-overloaded mice (109.6 6 6.0 and 87.8 6 3.7 nmol/g liver without and with nitroxyl probe, respectively, p , .001), although there was no significant difference in the liver iron content with or without the nitroxyl probe (data not shown). The effect of the nitroxyl probe on lipid peroxidation in vivo seemed to be additive with DF but not with Trolox. These facts strongly indicate that the nitroxyl probe must be involved in the free radical reactions responsible for lipid peroxidation caused by iron loading. The antioxidant activity of the nitroxyl probe was also confirmed with an in vitro experiment using liver homogenate (Table 3). Lipid peroxidation in the homogenate was induced by incubating it with 50 mM Fe(III)citrate and TBARs were suppressed by either DF (30 mM), carbamoyl-PROXYL (150 mM) or Trolox (150 mM). Co-incubation of DF or Trolox with the nitroxyl probe inhibited the TBARs relative to the control level, indicating the antioxidant effect of the nitroxyl probe. DISCUSSION
The in vivo signal decay of nitroxyl probe is reported to be related to physiological reducing capacity and free radical reactions in the body [31–39]. Intravenous injection of
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Fig. 3. Effect of desferrioxamine (DF) on iron excretion in iron overloaded mice (A) and the correlation between excreted iron content and suppression of enhanced signal decay by DF (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g) and a single dose of DF (0.19 mmol/g) was administered IP 10 min before (pre-), simultaneously with, or 20 or 50 min after iron loading. ESR signal of carbamoyl-PROXYL was observed 10, 30, and 60 min after iron loading, and the initial signal decay kinetic constant was obtained from the slope of a semi-logarithmic plot of signal intensity versus time after injection, as described in the legend of Fig. 1. The iron contents in kidney (u) and urine (h) were determined 90 min after iron loading. Results are presented as mean 6 SE of 4 –7 mice. Significant differences are *p , .05, **p , .01, ***p , .0001, compared with control and ##p , .05 compared with DF simultaneous treatment. The data used for correlation analysis were obtained from the difference of signal decay constant between before and after DF treatment, and excreted iron contents were measured with the same individual mice used for ESR experiment. The line was obtained by regression analysis (r 5 0.827, p , .001).
Fig. 4. In vivo effect of carbamoyl-PROXYL on lipid peroxidation of mice liver in the presence of DF (A) and Trolox (B). Mice were subcutaneously loaded with ferric citrate (0.2 mmol/g). Mice in the group of nitroxyl probe treatment (u) were intravenously injected with three doses of carbamoyl-PROXYL (0.75 mmol/g/dose) 10, 30, and 60 min after iron loading and then liver TBARs were estimated with its homogenate 90 min after iron loading as described in Methods. A single dose of DF (0.19 mmol/g) (A) or Trolox (0.4 mmol/g) (B) were given to iron overloaded mice 10 min before or 50 min after iron loading. Mice in the group of nitroxyl untreatment (■) were done as the same procedure as the nitroxyl treatment group without injection of the nitroxyl probe. Results are presented as mean 6 SE of 5–12 mice. Significant difference are *p , .05 and **p , .01 and between the groups indicated.
In vivo detection of Fe toxicity Table 3. In Vitro Antioxidant Activity of Carbamoyl-PROXYL on Lipid Preoxidation TBARs (nmol/ml) (a) Control Fe-citrate 50 mM 1 DF 30 mM 1 carbamoyl-PROXYL 150 mM 1 DF 30 mM and carbamoyl-PROXYL 150 mm (b) Control Fe-citrate 50 mM 1 Trolox 150 mM 1 carbamoyl-PROXYL 150 mM 1 Trolox 150 mM and carbamoyl-PROXYL 150 mM
0.74 6 0.35 17.50 6 0.007 6.77 6 0.001 9.41 6 0.027 0.50 6 0.014 0.68 6 0.033 12.60 6 0.001 2.66 6 0.000 4.10 6 0.001 0.76 6 0.006
Lipid peroxidation of liver homogenate was induced by incubation with 50 mM ferric citrate at 37°C for 1 h in the presence or absence of DF (30 mM) (a), Trolox (150 mM) (b), and carbamoyl-PROXYL (150 mM). TBARs were determined with the liver homogenate as described in Materials and Methods. Results are presented as mean 6 SE of duplicate.
nitroxyl probe in mice or rats produced triplet lines in the in vivo ESR measurement and the signal decay obeyed first order kinetics. The signal decay was increased by oxidative stress such as hyperoxia [32], ischemia-reperfusion [37], and x-irradiation [39], in disease models such as liver damage induced by CCl4 [32] and streptozotocin-induced diabetes [40]. In addition, the enhancement was suppressed by in vivo antioxidants [31,32,36,39]. In vitro experiments have also shown that nitroxyl probe reacts with various free radical species such as superoxide, hydroxyl, and peroxyl radicals [46]. The chain-breaking antioxidant property of water-soluble nitroxyl probe has been observed in liver microsome with NADPH [47,48]. This evidence strongly suggests that nitroxyl probe is very susceptible to free radical reactions involved in oxidative damage. The single dose of excess ferric citrate rapidly increased the in vivo signal decay, that was suppressed by both iron chelator and the chain-breaking antioxidant, suggesting a relationship between signal decay and free radical reactions catalyzed by iron. Indeed, the nitroxyl probe showed in vivo antioxidant activity by inhibiting lipid peroxidation (TBARs) in iron-overloaded mice, and this was confirmed by an in vitro experiment. Therefore, the enhanced signal decay produced by iron should be related to free-radical-mediated lipid peroxidation. It is widely believed that iron causes complications in several diseases by catalyzing free radical reactions that produce cell and tissue injuries [1]. Iron toxicity is not only involved in hemochromatosis, but also in cancer, ischemia, Parkinson’s Disease, and rheumatoid arthritis. Iron exists in various forms in biological systems, including heme iron, iron bound to proteins such as ferritin, hemosiderin, and transferrin, and “low molecular weight iron” such as iron
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ions attached to ATP, citrate, deoxyribose, and membrane lipids. The most interesting form of iron is the “low molecular weight pool,” because it is a catalytic iron responsible for toxic oxygen generation. Low molecular weight iron has been found in both the extracellular and intracellular compartments in iron-overloaded patients and animals [15–18], and also in many pathological conditions such as alcoholism, and ischemia-reperfusion [19–22]. In high iron overload, the capability of transferrin to maintain iron is exceeded, resulting in excessive amounts of low molecular weight iron in the blood compartment. Hepatocytes and macrophages rapidly take up the excess iron, resulting in expansion of the transit intracellular low molecular weight iron pool. Chronic and acute iron-overloaded animals have been used as models to investigate the mechanism of iron toxicity. Most of the animal experiments have evaluated the presumed effect of iron via free radical reactions by following changes in antioxidant enzyme activity, antioxidant status, and lipid peroxidation level [5–8]. However, there is little direct evidence that proves iron catalyzes free radical reactions and identifies the catalytic iron in vivo. The formation of iron-dependent oxygen free radicals by low molecular weight iron in biological systems is still controversial. Although “low molecular weight iron” has been identified as “Fenton-catalytic” iron, and the “bleomycin assay” has been developed to measure the availability of catalytic iron in biological samples [49], there is no direct proof that bleomycin-detectable iron and low molecular weight iron catalyze free radical reactions in vivo. Moreover, although the Fenton reaction is thought to produce hydroxyl radicals as the reactive species, it is also suggested that other species are formed, such as ferryl radical [12,14]. In addition, the reaction of iron complex with lipid hydroperoxides is an order of magnitude faster than the reaction with hydrogen peroxide [50,51], suggesting that, in biological systems, the direct reaction with lipid hydroperoxide is more likely to occur than that through the Fenton reaction. The only direct measurements involve in vivo spin trapping of methyl radicals, produced secondary from hydroxyl radicals with DMSO, and in vitro detection with X-band ESR spectroscopy [23–25]. This technique clearly demonstrated the in vivo generation of hydroxyl radical in acute and dietary iron-overloaded rats. Although the spintrapping technique suggest the significance of Fenton reaction in iron overloaded animals, the other reactions catalyzed by iron in vivo should be also considered. In addition, the association between catalytic activity and the form of iron in vivo is not well established. Therefore, direct and real-time determination of in vivo free radical reactions caused by iron is essential to better understanding the mechanism of its toxicity. The in vivo ESR measurement with a nitroxyl probe is a very suitable technique to investigate the mechanism of iron toxicity in vivo.
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Iron kinetic data suggest that low molecular weight iron is transiently present after a single injection of iron [52]. Desferrioxamine is an effective iron chelator. It readily chelates iron from the low molecular weight iron pool, and its complexes are rapidly excreted into the bile or urine, depending on the source of the iron [53,54]. Urinary iron appears to be derived from the “low molecular weight iron” in the plasma or the transit exchangeable pool in the liver, whereas biliary iron arises from ferritin degradation in the liver. Therefore, the experiments with DF should be able to estimate the time-dependence of the amount of low molecular weight iron. Because the sum amount of iron in the liver, kidney, and urine of iron-overloaded mice was no different in the DF-treated and non-treated groups, most of the iron-DF complexes were probably excreted into the urine. Although all of the DF treatments had an effect on signal decay, lipid peroxidation, and iron status, the effect was lower in the treatment 50 min after iron loading, suggesting the relatively short life of “chelatable iron” or “low molecular weight iron.” The signal decay suppressed by DF was very well correlated with the amount of urinary iron in iron overloaded mice, indicating that “chelatable iron” contributes to the enhanced signal decay. There was also a good correlation between the in vivo signal decay, iron content, and lipid peroxidation in the liver. These in vivo findings support the hypothesis that “low molecular weight iron” catalyzes free-radicalmediated lipid peroxidation. The enhanced signal decay caused by iron was suppressed by the chain-breaking antioxidant Trolox, and to a lesser degree by the hydroxyl radical scavenger DMSO. Iron preferentially stimulates peroxidation by lipid decomposition though reactions (1) and (2) [50], because there is usually plenty of hydroperoxide present in the cell membranes and plasma lipid component. Garnier-Suillerot et al. [51] reported that the kinetic constant of reaction (I) is 1.5 3 103 M21s21, that is higher than that of the Fenton reaction (76 M21s21). The reduced effect of DMSO, a hydroxyl radical scavenger, implies a smaller contribution of hydroxyl radical to the in vivo oxidative stress in the liver caused by iron. iron complex
2LOOH O ¡ LO 1 LO 2 1 H 2 O
(1)
LOOH 1 Fe 21 3 LO • 1 OH 2 1 Fe 31
(2)
Our results strongly indicate that in vivo ESR spectroscopy using a nitroxyl probe provides information about the catalytic form and activity of iron. They support the hypothesis that chelatable or low molecular weight iron plays a role in free radical reactions and lipid peroxidation. Iron should initiate lipid peroxidation pref-
erentially via reaction with lipid hydroperoxide and produce peroxyl and alkoxyl radicals. This hypothesis is supported by the effect of the chain-breaking antioxidant. The technique of in vivo ESR spectroscopy discussed here may be applied to evaluate new iron chelators and antioxidants for the therapy of iron-related diseases, such as hemochromatosis. Acknowledgements — This work was supported by a Grant-in-Aid for Research on Priority Areas, a Grant-in-Aid for Co-operative Research, a Grant-in-Aid for Developmental Scientific Research, a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan, by the Neito Foundation, and by the Cosmetology Research Foundation.
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ABBREVIATIONS
BHT— butyl hydroxy toluene Carbamoyl-PROXYL—3-Carbamoyl-2,2,5,5,5-tetramethylpyrrolidine-1-oxyl DF— desferrioxamine DMSO— dimethyl sulfoxide TBARs—thiobarbituric acid reactive substances