Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Free Radical Biology & Medicine, Vol. 28, No. 4, pp. 854 – 859, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00162-3

Forum: Oxidative Stress Status NONINVASIVE STUDY OF RADIATION-INDUCED OXIDATIVE DAMAGE USING IN VIVO ELECTRON SPIN RESONANCE YURI MIURA*

and

TOSHIHIKO OZAWA†

*Department of Biochemistry and Isotopes, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan and †Department of Bioregulation Research, National Institute of Radiological Sciences, Chiba, Japan (Received 30 September 1999; Revised 29 December 1999; Accepted 10 January 2000)

Abstract—Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body. © 2000 Elsevier Science Inc. Keywords—In vivo ESR, Oxidative stress, Nitroxyl radical, ROS, Spin probe, Signal decay, Redox, Free radicals

INTRODUCTION

tremely low concentrations in tissue and organs and because their lifetimes are extremely short, indirect detection employing spin trapping agents or spin probes is more useful. The most widely used spin probe is a nitroxyl radical, which is relatively stable at room temperature and has low toxicity to organisms (Fig. 1). Nitroxyl radical is not stable in biological systems, however. In hepatic microsomes and various cultured cells, the reduction and reoxidation of nitroxyl compounds have been reported since the 1970s, suggesting that various biomolecules and enzymes such as cytochrome P-450, cytochrome P-450 reductase, mitochondrial electron transport systems, and ascorbic acid, are involved in the redox reaction of nitroxyl. Furthermore, it has become apparent that oxygen concentration, antioxidant content, and oxygen free radicals influence the rate of the redox reaction of nitroxyl in biological systems. Therefore, in in vitro or cell-level experiments, a nitroxyl compound may be used as a sensitive redox indicator by monitoring its redox reaction. In whole-body experiments, there have been several recent reports noting the redox change in organisms in pathophysiological conditions such as oxidative stress and examining the effects on the redox reaction of nitroxyl in vivo. Here, we review the recent in vivo ESR studies on the redox reaction of nitroxyl and discuss the

As it has become apparent that oxygen free radicals are involved in numerous pathophysiological conditions, there is a growing interest in the in vivo detection of free radicals by electron spin resonance (ESR). ESR was developed as an in vivo detector of bioradicals in the late 1980s. Because bioradicals such as nitric oxide, superoxide radical, and hydroxyl radical are present in exYuri Miura graduated with a degree in pharmaceutical sciences from the University of Tokyo. She was a Research Assistant at the School of Pharmaceutical Sciences, Showa University, and received her Ph.D. degree from the University of Tokyo in 1994. She completed a postdoctoral fellowship with Dr. Toshihiko Ozawa at the National Institute of Radiological Sciences. She is currently a Research Scientist at Tokyo Metropolitan Institute of Gerontology. Her research interests include radiation biology of neuronal and glial cells. Toshihiko Ozawa graduated with a degree in pharmaceutical sciences from the University of Tokyo. In 1974, he received his Ph.D. degree from the same university and performed his postdoctoral work with Dr. James P. Collman at Stanford University before becoming a Senior Researcher at the National Institute of Radiological Sciences. He was then a Section Head, and in 1993, he assumed the position of Director of the Department of Bioregulation Research. He is also a Visiting Professor at the Graduate School of Natural Sciences at Chiba University. His research interests include in vivo detection and electron spin resonance imaging of active oxygens and free radicals. Address correspondence to: Toshihiko Ozawa, Department of Bioregulation Research, National Institute of Radiological Sciences, Chiba 263-8555, Japan; Tel: ⫹81 (43) 206 3120; Fax: ⫹81 (43) 255 6819; E-Mail: [email protected] 854

In vivo ESR study of oxidative stress

Fig. 1. Chemical structures of nitroxyl compounds.

possibility of applying in vivo ESR to the in vivo noninvasive study of oxidative damage. PHARMACOKINETICS OF NITROXYL RADICALS INJECTED TO WHOLE BODY

In in vivo ESR measurement, the signal intensity of a nitroxyl radical decreases with time after injection. The pharmacokinetics of a nitroxyl compound (carbamoylPROXYL) using L-band (approximately 1 GHz microwave) ESR was first reported by Berliner and Wan [1]. They detected the nitroxyl signal of carbamoylPROXYL and measured the signal disappearance in rat tails. Since then, a several groups have reported spectra and the signal decay of nitroxyl radical in the abdomen or head of living animals by L-band ESR [2–7]. The signal decay of nitroxyl in the whole body is ascribed to two factors: one is the metabolism of nitroxyl to a diamagnetic molecule and the other is the diffusion and excretion from the measured region to other organs. In vitro, there are many reports of the reduction of nitroxyl radicals to the corresponding hydroxylamine in microsomes, mitochondria, and whole cells. In vivo, nitroxyl compounds also seem to be reduced to hydroxylamine, based on the fact that the signal intensity of the collected blood was recovered by the addition of potassium ferricyanide, which oxidized hydroxylamine to nitroxyl radical [8]. Takeshita et al. [9] extracted the metabolite of hydroxy-TEMPO from mouse lung and identified it as its hydroxylamine by means of thin-layer chromatography. The reduction rates of nitroxyl depend on the chemical structure of nitroxyl compounds. Kom-

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arov and Lai [10] have measured the in vivo reduction kinetics of about 20 different nitroxyl compounds by S-band (3.5 GHz microwave) ESR. They examined the effect of the chemical structures of nitroxyl compounds on the half-life of in vivo reduction, showing that pyrrolidine nitroxyl was more resistant to cellular metabolism in vivo than piperidine nitroxyl. In vivo, the reducing enzymes and ascorbate should be involved in the reduction of a nitroxyl radical similar to what occurs in vitro. Vianello et al. [11] calculated the ascorbate contribution to various nitroxyl removals on the basis of the ascorbate concentration in organs and the second-order kinetic constants of nitroxyl reduction measured in phosphate-buffered saline. They speculated that the disappearance of piperidine nitroxyl in the brain was controlled mainly by ascorbate, although that of pyrrolidine nitroxyl was not. On the other hand, Bacic et al. [3] have reported the detection of an increase in nitroxyl signal in the bladder of a mouse injected with Cat1 using L-band ESR. Takechi et al. [12] examined the urine of rats that were administered 3-carboxy-PROXYL, and their results suggested that parent nitroxyl and its hydroxylamine were excreted into the urine 2 to 10 h after administration. These data suggest that water-soluble nitroxyl compounds were excreted into the urine. EFFECTS OF VARIOUS TYPES OF OXIDATIVE STRESS ON SIGNAL DECAY RATES OF NITROXYL RADICALS

Because various types of oxidative stress are known to change the redox state and metabolic capacity of organisms, it is expected that they would affect the pharmacokinetics of nitroxyl radical in vivo. Thus, the effects of oxidative stress or the administration of various drugs were studied by examining the signal decay rates of various nitroxyls [8,13–21] (Table 1). The results are divided into two categories depending on whether the treatment caused an increase or decrease in the signal decay rate of nitroxyl radicals. In cases in which signal decay was inhibited, the results were attributed to a decrease in the reducing capacity of the organism [8,20, 21]. In contrast, treatment with idebenone or chronic administration of vitamin C was concluded to enhance the radical reducing ability in the living body, resulting in an increase in the decay rates of nitroxyl [18,19]. In cases of oxidative stress such as ischemia-reperfusion, hyperoxia, diabetes, iron overload, and CCl4 administration [13–17], it was speculated that a free radical reaction occurring during the oxidative damage may have been involved in the enhancement. This was because the enhancement was suppressed by in vivo antioxidants and because in vitro nitroxyl radical reacts with various ROS such as superoxide, hydroxyl radicals, and peroxyl rad-

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MIURA and OZAWA Table 1. Effects of Various Treatments in Animals on the Rates of Signal Decay of Nitroxyl Radicals

Effect Increase Increase Increase Increase Increase Increase Increase Decrease Decrease Decrease

Treatment

Measured region

Spin probe

Experimental condition

Reference

Ischemia-reperfusion Hyperoxia Diabetes Iron overload CCl4 Idebenone Vitamin C Aging Seizure CCl4

Femoral Abdomen Abdomen Abdomen Abdomen Head Head Head Head Hepatic and pelvic domain

Amino-TEMPO Carbamoyl-PROXYL Carbamoyl-PROXYL Carbamoyl-PROXYL Carbamoyl-PROXYL Carbamoyl-PROXYL Hydroxy-TEMPO Carbamoyl-PROXYL Carbamoyl-PROXYL Hydroxy-TEMPO

Occlusion Exposed to 80% O2 and 20% N2 Streptozotocin-induced Subcutaneously loaded with ferric-citrate Oral administration Intracerebroventricular injection Vitamin C–containing food 6, 30, and 39 month old mice Kainic acid–induced Intraperitoneal administration

[13] [14] [15] [16] [17] [18] [19] [8] [20] [21]

icals, leading to the signal disappearance. The results of CCl4 administration were different between the report of Inaba et al. [21] and that of Utsumi et al. [17]. Inaba et al. [21] injected CCl4 intraperitoneally and measured in vivo ESR 48 h after injection, and Utsumi et al. [17] used oral administration and measured in vivo ESR 30 min after injection. CCl4 injected into an organism is metabolized by cytochrome P-450 in liver microsomes, which accompanies the generation of ROS, causing hepatic damage and a decrease in metabolic capacity. It seems that there is a change in the degree of the damage between 30 min and 48 h after administration. Therefore, these results suggest that different stages of oxidative stress should yield different effects on the signal decay rate of nitroxyl radical. EFFECTS OF RADIATION ON SIGNAL DECAY RATES OF CARBAMOYL-PROXYL IN THE ABDOMENS OF MICE

Radiation produces various ROS such as hydroxyl radicals, superoxide, and hydrogen peroxide in the whole body not only directly but also indirectly through a subsequent free radical reaction and inflammation, resulting in a change in the redox status of the organism. We have examined the effects of radiation on the decay rate of a nitroxyl radical (carbamoyl-PROXYL) using L-band ESR [22].

In experiments in noncysteamine-treated mice, the mice were separated into six groups. Groups 1 and 2 were treated by sham irradiation as a control. Groups 3 and 4 were treated by X-irradiation at a dose of 7.5 Gy, which is approximately the LD50/30 of mice, and groups 5 and 6 were treated by 15 Gy irradiation. In vivo ESR measurement was performed 1 h after irradiation in groups 1, 3, and 5, and 4 or 5 d after irradiation in groups 2, 4, and 6. In cysteamine-treated mice, cysteamine was injected into the mice intraperitoneally 20 min before irradiation (2.0 mM/kg). Whole-body irradiation was performed at a dose rate of approximately 0.6 Gy/min. Sham irradiation of the controls included comparable immobilization in the same irradiation chamber. Anesthetic was not administered to either irradiated or shamirradiated mice. The in vivo ESR spectra of the nitroxyl radical were measured as follows. A mouse was anesthetized using pentobarbital and placed in a loop-gap resonator. The solution of carbamoyl-PROXYL was injected into the tail vein, and ESR spectra were measured in the upper abdomen of the mouse repeatedly beginning immediately after injection. The rate constants of the signal decay of nitroxyl were calculated from the signal decay curves, which were determined from semilogarithmic plots of the peak heights of the ESR signal at the lower magnetic field. Table 2 summarizes the kinetic constants of signal decay in the abdomens of the mice. One hour after

Table 2. Radiation Effects on the Signal Decay Rates (Gy/min) of Carbamoyl-PROXYL in the Abdomens of Mice 1 h After irradiation

0 Gy 7.5 Gy 15.0 Gy

4 or 5 d After *irradiation

Without cysteamine

With cysteamine

Without cysteamine

With cysteamine

0.109 ⫾ 0.015 0.130 ⫾ 0.029 0.145 ⫾ 0.021*

0.102 ⫾ 0.010 — 0.100 ⫾ 0.005

0.125 ⫾ 0.031 0.165 ⫾ 0.029 0.075 ⫾ 0.008†

— 0.138 ⫾ 0.004 —

* p ⬍ .001, different from that 1 h after 0 Gy (without cysteamine). † p ⬍ .05, different from that 4 or 5 d after 0 Gy (without cysteamine).

In vivo ESR study of oxidative stress

irradiation, the signal decay rates increased by 15 Gy irradiation; 5 d after irradiation, these rates significantly decreased by 15 Gy irradiation. These data suggest that there are at least two factors that affect the signal decay rate of nitroxyl: one causing enhancement and another causing inhibition. Five days after irradiation, the mice exposed to 15 Gy irradiation were severely damaged; about half of the mice died within 5 d. The factor causing inhibition was believed to be the degeneration of the systemic condition, including metabolic and excretive capacities, as the result of high-dose irradiation. To study the factor causing enhancement, we examined the effect of cysteamine, which is a radical scavenger and radioprotector, on the enhancement of the signal decay rate in nitroxyl (Table 1). It seemed that preadministration of cysteamine significantly suppressed the enhancement of the signal decay rate of nitroxyl as a result of X-irradiation. Other radioprotectors such as 5-HT, WR2721, hydroxy-TEMPO, IL-1␤, and SCF also suppressed the enhancement of the signal decay rate of nitroxyl at the appropriate doses.

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EFFECT OF RADIATION ON SIGNAL DECAY RATES OF MCPROXYL IN THE HEADS OF MICE

The spin probe injected into peripheral blood cannot be distributed to brain tissue because of the blood brainbarrier (BBB). Thus, we synthesized BBB-permeable spin probe, and radiation damage to the brain was examined using in vivo ESR [23]. MCPROXYL was more lipophilic than carbamoylPROXYL and well distributed in brain tissue after intravenous injection. The signal decay rate of nitroxyl radical in the head region decreased 1 h after irradiation unlike that of carbamoyl-PROXYL in the upper abdomen. We examined the effect of radiation on the reducing activity of nitroxyl in the brain homogenate and the content of ascorbic acid in the brain, and the results showed that the reducing capacity was not decreased 1 h after irradiation. Although the biological mechanism of the radiation effect on MCPROXYL disappearance remains unclear, it is possible that the BBB-permeable spin probe might provide some information on radiation damage in the brain.

BIOLOGICAL MECHANISM OF THE ENHANCEMENT OF NITROXYL DECAY IN THE WHOLE BODY

TOPICS

What is the factor causing enhancement in this case? There are two possibilities: the induction of reducing capacity by oxidative stress and the participation of free radical reaction as a result of X-irradiation. We examined the activity of reducing enzymes (cytochrome P-450 and cytochrome P-450 reductase) and antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase) as well as the contents of endogenous antioxidants (vitamins E and C) under the present conditions. The results indicated that neither the activities of the reducing and antioxidative enzymes nor the contents of endogenous antioxidants increased 1 h after irradiation, suggesting that the reducing capacities in the mice were not induced 1 h after irradiation. Accordingly, free radical reactions in tissue caused by X-irradiation might be involved in the enhancement of the signal decay rate of nitroxyl. Although the lifetimes of primarily formed ROS are quite short as a result of X-irradiation, they should subsequently cause biological and chemical chain reactions, which, in turn, would produce fresh ROS. The fact that the radical scavenger cysteamine suppressed the enhancement seems to support the hypothesis that free radical reaction induced by X-irradiation participates in the enhancement of nitroxyl decay similar to what occurs in other types of oxidative stress [13–17]. Because in vivo ESR study is a whole-body experiment, however, physiological factors that affect the rate of tissue distribution or excretion of nitroxyl (e.g., blood pressure, blood flow rate, body temperature) cannot be ruled out.

The analysis of in vivo ESR data is quite difficult because of the many factors affecting the signal decay rate of nitroxyl radicals in the whole body. Nevertheless, we can say for certain that the signal decay rate of nitroxyl radicals in vivo reflects the pathophysiological and/or physiological state of a living body. Therefore, we believe that in vivo ESR can be used for clinical applications so as to probe the change of the signal decay rate in nitroxyl. Recently, there have been many in vivo ESR studies aiming to improve in vivo imaging. Nicholson et al. [24] have reported the in vivo imaging of nitroxyl clearance using a LODESR imaging apparatus to demonstrate that carboxy-PROXYL injected into rats shifted from the liver to the kidneys with time after injection. Yokoyama et al. [25] have reported ESR imaging for the signal decay of nitroxyl in the brains of rats. They used MCPROXYL, a BBB-permeable spin probe, and analyzed the effect of kainic acid–induced seizures on the signal decay rates in the hippocampus and cerebral cortex, showing that the half-life of nitroxyl radicals was significantly prolonged in the hippocampus but not in the cerebral cortex by kainic acid–induced seizure. In vivo imaging of the signal decay rate of nitroxyl should provide more information on the pathophysiology of the living body. Conversely, it is also necessary to develop the spin probe, whose spectrum yields pathophysiological or physiological information on organisms. Here, we sum-

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marize the recent reports on such spin probes. Gallez et al. [26] have developed a pH-sensitive nitroxyl compound, which is manifested in the ESR spectrum as a decrease in hyperfine coupling constant with pH-induced change, and Sotgiu et al. [27] have performed pH-sensitive imaging using this nitroxyl. Dragutan et al. [28] also synthesized pH-sensitive spin probes. Using these spin probes, in vivo ESR can provide a noninvasive technique for monitoring pH in tissue or organs. Furthermore, Yamaguchi et al. [29] synthesized spin-labeled triglyceride (SL-TG) and analyzed in vivo ESR spectra of lipid emulsion containing SL-TG in the chests of mice. Immediately after administration, ESR signal derived from lipid particles was observed; after that, ESR signal derived from free and immobilized fatty acids to which lipoprotein lipase in the blood hydrolyzed lipid particles was superimposed on the spectra. These investigators demonstrated that in vivo ESR can determine the pharmacokinetics of lipid emulsion in a noninvasive fashion, suggesting that oxidative damage of lipoproteins or blood cells in the living body may be analyzed by this method.

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] CONCLUSIONS [17]

Nitroxyl radicals injected into an organism indicate the disappearance of signal intensity of in vivo ESR. It is shown that the signal decay rates of nitroxyl depend on various types of oxidative stress, including X-irradiation. Thus, in vivo ESR could provide a noninvasive technique for the study of oxidative stress in a living body. Acknowledgements — This study was supported in part by a Grant-inAid for Scientific Research (No. 10357021) from the Ministry of Education, Science, Sports, and Culture of Japan, and by a Grant from the Cosmetology Research Foundation.

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In vivo ESR study of oxidative stress of the pH inside the gut by using pH-sensitive nitroxides. An in vivo EPR study. Magn. Reson. Med. 36:694 – 697; 1996. [27] Sotgiu, A.; Mader, K.; Placidi, G.; Colacicchi, S.; Ursini, C. L.; Alecci, M. pH-sensitive imaging by low-frequency EPR: a model study for biological applications. Phys. Med. Biol. 43:1921–1930; 1998. [28] Dragutan, H.; Caragheorgheopol, A.; Chiralen, F.; Mehlhorn, R. J. New amino-nitroxide spin labels. Bioorg. Med. Chem. 4:1577–1583; 1996. [29] Yamaguchi, T.; Itai, S.; Hayashi, H.; Soda, S.; Hamada, A.; Utsumi, H. In vivo ESR studies on pharmacokinetics and metabolism of parenteral lipid emulsion in living mice. Pharm. Res. 13:729 –733; 1996. ABBREVIATIONS

ESR— electron spin resonance ROS—reactive oxygen species carbamoyl-PROXYL—3-carbamoyl-2,2,5,5-tetramethyl pyrrolidine-1-oxyl

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hydroxy-TEMPO— 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl Cat1— 4-trimethylamino-2,2,6,6-tetramethyl piperidine1-oxyl carboxy-PROXYL—3-carboxy-2,2,5,5-tetramethyl pyrrolidine-1-oxyl MCPROXYL—3-methoxymethyl-2,2,5,5-tetramethyl pyrrolidine-1-oxyl CCl4— carbon tetrachloride LD50/30—50% lethal dose for 30 d 5-HT—5-hydroxytryptamine WR2721—S-2-(3-aminopropylamino)ethylphosphorothioic acid IL-1␤—interleukin 1␤ SCF—stem cell factors LODESR—longitudinally detected ESR