Free Radical Biology & Medicine 42 (2007) 44 – 51
`
`www.elsevier.com/locate/freeradbiomed
`
`Original Contribution
`Oxalomalate regulates ionizing radiation-induced apoptosis in mice
`Jin Hyup Lee, Jeen-Woo Park ⁎
`
`School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea
`
`Received 12 June 2006; revised 25 August 2006; accepted 15 September 2006
`Available online 23 September 2006
`
`Abstract
`
`Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently,
`we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of
`mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate
`that modulation of IDPm activity in the kidneys of mice regulates ionizing radiation-induced apoptosis. When oxalomalate, a competitive inhibitor
`of IDPm, was administered to mice, inhibition of IDPm and enhanced susceptibility of apoptosis reflected by DNA fragmentation, the changes in
`mitochondria function, and the modulation of apoptotic marker proteins were observed upon exposure to 2 Gy of γ-irradiation. We also observed a
`significant difference in the mitochondrial redox status between the kidneys of the control and the oxalomalate-administered mice. This study
`indicates that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, presumably, through acting as an
`antioxidant enzyme.
`© 2006 Elsevier Inc. All rights reserved.
`
`Keywords: Ionizing radiation; IDPm; Oxalomalate; Apoptosis; Redox status
`
`Introduction
`
`Radiation therapy has been commonly used for the treatment
`of tumors. Ionizing radiation has been shown to generate
`reactive oxygen species (ROS) in a variety of cells [1]. When
`water, the most abundant intracellular material, is exposed to
`ionizing radiation, decomposition reactions occur and a variety
`of ROS,
`including superoxide, hydroxyl
`radicals, singlet
`oxygen, and hydrogen peroxide, are generated [2]. The
`secondary radicals formed by the interaction of hydroxyl
`radicals with organic molecules may also be of importance
`[1,2]. These ROS have the potential to damage critical cellular
`
`Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutase;
`ICDHs, isocitrate dehydrogenases; IDPm, mitochondrial ICDH; DNPH, 2,4-
`dinitrophenylhydrazine; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TRITC,
`tetramethylrhodamine isothiocyanate; DHR, dihydrorhodamine; PARP, poly
`(ADP-ribose) polymerase; JC-1, 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimi-
`dazolcarbocyanine iodide; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine; MDA,
`malondialdehyde; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
`electrophoresis.
`⁎ Corresponding author. Fax: +82 53 943 2762.
`E-mail address: parkjw@knu.ac.kr (J.-W. Park).
`
`0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
`doi:10.1016/j.freeradbiomed.2006.09.016
`
`components such as DNA, proteins, and lipids and eventually
`result in physical and chemical damage to tissues that may lead
`to cell death or neoplastic transformation [3]. In many cases,
`ionizing radiation-induced cell death has been identified as
`apoptosis [4].
`Biological systems have evolved an effective and compli-
`cated network of defense mechanisms which enable cells to
`cope with lethal oxidative environments. These defense
`mechanisms involve antioxidant enzymes, such as superoxide
`− to
`dismutase (SOD), which catalyzes the dismutation of O2
`H2O2 and O2 [5], catalase, and peroxidases which remove
`hydrogen peroxide and hydroperoxides [6]. Since ROS appear
`to be mediators of the apoptosis by ionizing radiation [7],
`factors including antioxidant enzymes that regulate the fate of
`such species may be of great importance in the protection of
`cells against ionizing radiation-induced cell death.
`The isocitrate dehydrogenases (ICDHs, EC1.1.1.41 and
`EC1.1.1.42) catalyze oxidative decarboxylation of isocitrate to
`α-ketoglutarate and require either NAD+ or NADP+, producing
`NADH and NADPH, respectively [8]. NADPH is an essential
`reducing equivalent for the regeneration of reduced glutathione
`(GSH) by glutathione reductase and for the activity of NADPH-
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`45
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`dependent thioredoxin system [9,10], both are important in the
`protection of cells from oxidative damage. Therefore, ICDH
`may play an antioxidant role during oxidative stress. We
`recently reported that mitochondrial ICDH (IDPm) is involved
`in the supply of NADPH needed for GSH production against
`mitochondrial oxidative damage [11].
`In the present report, we demonstrate that the modulation of
`IDPm activity by oxalomalate in the kidneys of mice regulates
`apoptosis induced by γ-irradiation. Oxalomalate, a tricarboxylic
`acid (α-hydroxy-β-oxalosuccinic acid) formed in vitro and in
`vivo by condensation of oxaloacetate and glyoxylate, has been
`known to be a potent inhibitor of IDPm [12]. The results suggest
`that IDPm may play an important role in regulating the
`apoptosis induced by ionizing radiation, presumably, through
`acting as an antioxidant enzyme.
`
`Materials and methods
`
`Materials
`
`β-NADP+, isocitrate, 2,4-dinitrophenylhydrazine (DNPH),
`5,5-dithiobis(2-nitrobenzoic acid) (DTNB), xylenol orange,
`propidium iodide (PI), oxalomalate, avidin-conjugated tetra-
`methylrhodamine isothiocyanate (TRITC), and anti-rabbit IgG-
`conjugated TRITC secondary antibody were obtained from
`Sigma Chemical Co. (St. Louis, MO). 5,5,6,6-Tetrachloro-
`1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-1), and
`dihydrorhodamine (DHR) 123 were purchased from Molecular
`Probes (Eugene, OR). Electrophoreses reagents and the Bio-
`Rad protein assay kit were purchased from Bio-Rad (Hercules,
`CA). Antibodies against Bcl-2, Bax, cleaved caspase-3, cleaved
`poly(ADP-ribose) polymerase (PARP), cytochrome c, Noxa,
`PUMA, p21, and p53 were purchased from Santa Cruz (Santa
`Cruz, CA) or Cell Signaling (Beverly, MA).
`
`Mice
`
`Six- to 8-week-old female C57BL/6 mice were purchased
`from the National Cancer Institute (Frederick, MD) and kept in
`the oncology animal facility of the Johns Hopkins Hospital
`(Baltimore, MD). All animal procedures were performed
`according to approved protocols and in accordance with
`recommendations for the proper use and care of laboratory
`animals.
`
`Animal treatment and whole-body irradiation
`
`Two groups of 15 C57BL/6 mice each received either
`oxalomalate or 0.9% NaCl. Solutions of oxalomalate were
`freshly prepared in 0.9% NaCl and administered before
`irradiation at a dose of 25 mg/kg in volumes equivalent to 1%
`of each animal's weight once daily for 10 days. Control mice
`were given 0.9% NaCl, and all injections were administered ip.
`After irradiation with a 137Cs source at a dose rate of 1 Gy/min,
`the mice were returned to climate-controlled cages for further
`experiment. Mice were sacrificed by a cervical dislocation at
`7 days postirradiation.
`
`Tissue dissociation
`
`Dissociation of kidney tissue was performed using a petri
`dish, tweezers, and a fresh scalpel blade. Dissociated tissue was
`prevented from drying out by covering it with a few drops of
`PBS. During dissociation, the kidney epithelial cell suspension
`was aspirated repeatedly through a 20-gauge needle into a
`syringe, and consequently injected through a cell strainer into a
`50-ml tube to retain clumps. Suspensions were centrifuged and
`resuspended with a defined amount of PBS, and final cell
`number was counted from aliquots.
`
`Measurement of IDPm activity
`
`For the preparation of mitochondrial fraction from kidney
`tissue, the tissue portions were homogenized with a Dounce
`homogenizer in sucrose buffer (0.32 M sucrose, 10 mM Tris-Cl,
`pH 7.4). Tissue homogenate was centrifuged at 1000 g for 5 min
`and the supernatants were further centrifuged at 15,000 g for
`30 min. The precipitates were washed twice with sucrose buffer
`to collect mitochondria pellet. The mitochondrial pellets were
`resuspended in 1× PBS containing 0.1% Triton X-100,
`disrupted by ultrasonication (4710 Series, Cole-Palmer, Chi-
`cago, IL) twice at 40% of maximum setting for 10 s, and
`centrifuged at 15,000 g for 30 min. The supernatants were used
`to measure the activity of IDPm. The protein levels were
`determined by the method of Bradford using reagents purchased
`from Bio-Rad.
`
`Determination of apoptotic cell death in tissue
`
`tissue, unfixed, dissociated
`Following dissociation of
`epithelial cells from kidney tissue were washed again in
`PBS and then slowly injected into cold methanol (4 °C, 100%)
`to a final concentration of 70% methanol, and kept at 4 °C
`until staining. Methanol-fixed cell suspensions were stained by
`an indirect
`immunofluorescence technique. Detection of
`apoptotic cells in tissue was performed using PE-conjugated
`rabbit anti-cleaved caspase-3 IgG (BD Bioscience, San Diego,
`CA) according to the vendor's protocol after gating a cell
`population. The percentage of apoptotic cells was analyzed by
`flow cytometry.
`
`DNA fragmentation
`
`DNA fragmentation was evaluated by TdT-mediated dUTP
`nick-end labeling (TUNEL) assay. After the TUNEL reaction,
`cells or histologic paraffin-embedded sections were analyzed by
`fluorescence microscopy. The green fluorescence of fluorescein
`isothiocyanate (FITC) was recorded with excitation at 488 nm
`through a 515-nm bandpass, together with the transmission
`image.
`
`Cellular oxidative damage
`
`Intracellular hydrogen peroxide concentrations were deter-
`mined using a ferric-sensitive dye, xylenol orange, as described
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`subsequently subjected to immunoblot analysis using appro-
`priate antibodies. Immunoreactive antigen was then recog-
`nized by using horseradish peroxidase-labeled anti-rabbit IgG
`and an enhanced chemiluminescence detection kit (Amer-
`sham Pharmacia Biotech).
`
`Statistical analysis
`
`The difference between two mean values was analyzed by
`Student's t test and was considered to be statistically significant
`when p < 0.05.
`
`Results
`
`IDPm activity
`
`To study the relationship between IDPm activity and
`ionizing radiation-induced apoptotic cell death, mice were
`administered 25 mg/kg oxalomalate, a competitive inhibitor of
`IDPm, once daily for 10 days. When mice were unexposed
`and exposed to 2 Gy of γ irradiation, the enzyme activity of
`IDPm in the kidneys of oxalomalate-treated mice was
`decreased about 20% compared with that of the control mice
`(Fig. 1). Because cellular antioxidants act
`in a concerted
`manner as a team, it is important to investigate whether the
`modulation of IDPm activity caused concomitant alterations in
`the activity of other major antioxidant enzymes. The reduced
`activity of IDPm in the kidneys of the oxalomalate-treated
`mice did not significantly alter
`the activities of other
`antioxidant enzymes such as SOD, catalase, glucose 6-
`phosphate dehydrogenase, glutathione peroxidase, and glu-
`tathione reductase (data not shown), suggesting that
`the
`reduction of IDPm activity did not affect
`the activities of
`other enzymes involved in antioxidation.
`
`Apoptosis determination
`
`The effects of ionizing radiation and IDPm activity on the
`cellular markers of apoptosis were studied to determine
`whether these would correlate with changes in the apoptotic
`pathways. As shown in Fig. 2A,
`the percentage of active
`
`[13]. Thiobarbituric acid-reactive substances (TBARS) were
`determined as an independent measurement of lipid peroxida-
`tion. The tissue homogenates (500 μl) were mixed with 1 ml
`TBA solution (0.375% thiobarbituric acid in 0.25 N HCl
`containing 15% (w/w) trichloroacetic acid and heated at 100 °C
`for 15 min. Then the reaction was stopped on ice, and the
`absorbance was measured at 535 nm [14]. The protein carbonyl
`content was determined spectrophotometrically using the
`DNPH-labeling procedure as described [15]. 8-Hydroxy-2-
`deoxyguanosine (8-OH-dG)
`levels of kidney tissue were
`estimated by using a fluorescent binding assay as described
`by Struthers et al. [16]. DNA damage was visualized with
`avidin-conjugated TRITC (1:200 dilution)
`for
`fluorescent
`microscopy with 540 nm excitation and 588 nm emission.
`
`Mitochondrial redox staus and damage
`
`NADPH was measured using the enzymatic cycling
`method as described by Zerez et al. [17] and expressed as
`the ratio of NADPH to the total NADP pool. The
`concentration of total glutathione was determined by the rate
`of
`formation of 5-thio-2-nitrobenzoic acid at 412 nm
`(ε = 1.36 × 104 M−1 cm−1) as described by Akerboom and
`Sies [18], and oxidized glutathione (GSSG) was measured by
`the DTNB-GSSG reductase recycling assay after treating GSH
`with 2-vinylpyridine. The mitochondrial membrane potential
`(Δφm) was semiquantitatively determined using the mitochon-
`drial-specific lipophilic fluorescent cationic probe JC-1 as
`described [19]. Following dissociation of
`tissue, unfixed,
`dissociated cells were washed and resuspended in PBS,
`supplemented with 10 μg/ml JC-1. Then cells were incubated
`for 15 min at room temperature in the dark, washed, and
`resuspended in PBS for immediate flow cytometry using a
`FACScan cytometer. Photomultiplier settings were adjusted to
`detect JC-1 monomers and J-aggregate fluorescence on the
`FL1 (530 nm) and FL2 (585 nm) detectors, respectively. The
`fluorescence ratio at those wavelengths was used to monitor
`changes in mitochondrial membrane potential. Fragmented
`cells and debris were excluded from measurements by gating
`the remaining intact cells in a forward and side scatter
`analysis. Data were analyzed using the CellQuest software
`(Becton Dickinson)
`to calculate the percentage of JC-1-
`positive cells. To evaluate the levels of mitochondrial ROS
`dissociated cells from tissue in PBS were incubated for
`20 min at 37 °C with 5 μM DHR 123 and cells were washed
`and resuspended in complete growth media, and ionizing
`radiation was applied to the cells. The cells were then
`incubated for an additional 40 min. FACS was used for
`fluorescence intensity quantification. Intracellular ATP levels
`were determined by using luciferin-luciferase as described
`[20]. Light emission was quantitated in a Turner Designs TD
`20/20 luminometer (Stratec Biomedical Systems, Germany).
`
`Immunoblot analysis
`
`Proteins were separated on 10–12.5% SDS-polyacryla-
`mide gel,
`transferred to nitrocellulose membranes, and
`
`Fig. 1. Activity of IDPm in the kidneys of the control and the oxalomalate-
`administered mice unexposed and exposed to γ irradiation. Each value
`represents the mean ± SD of samples from five animals. *p < 0.01 vs unirradiated
`control mice. #p < 0.01 vs irradiated control mice.
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`47
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`tion were evaluated. It is well established that oxidative stress
`in various cells usually leads to accumulation of potent,
`cytotoxic lipid peroxides such as malondialdehyde (MDA) and
`4-hydroxynonenal [21]. Exposure of 2 Gy of γ-irradiation
`increased the level of MDA in the kidney of the control mice;
`however, the increase in the MDA content of the kidneys of
`the oxalomalate-administered mice was significantly higher
`than that of the control mice (Fig. 3B). To determine whether
`reduced IDPm activity enhanced the sensitivity of protein
`damage, we performed carbonyl content measurements for
`protein oxidation after exposure to ionizing radiation. When 2
`Gy of ionizing radiation was exposed,
`the kidneys of the
`oxalomalate-administered mice elicited an approximately 4.3-
`fold increase of carbonyl groups, compared to the unirradiated
`mice. Although the carbonyl content of the control mice also
`increased with irradiation, the increase was significantly lower
`than that of the oxalomalate-administered mice (Fig. 3C). The
`reaction of intracellular ROS with DNA resulted in numerous
`forms of base damage, and 8-OH-dG is one of the most
`abundant and most studied lesions generated. 8-OH-dG has
`been used as an indicator of oxidative DNA damage in vivo
`and in vitro [22]. Recently, it has been shown that 8-OH-dG
`level is specifically measured by a fluorescent binding assay
`using avidin-conjugated TRITC [16]. As shown in Fig. 3E, the
`fluorescent intensity which reflects the endogenous levels of
`8-OH-dG in DNA was significantly increased in the kidneys of
`the oxalomalate-administered mice compared to control mice
`on exposure to ionizing radiation. These results indicate that
`IDPm appears to protect mice from oxidative damage caused
`by ionizing radiation.
`
`Mitochondrial redox status and damage
`
`One important parameter of GSH metabolism is the ratio of
`GSSG/total GSH (GSHt) which may reflect the efficiency of
`GSH turnover. When the mice were exposed to 2 Gy of
`γ-irradiation, the ratio of mitochondrial [GSSG]/[GSHt] was
`significantly higher
`in the kidneys of
`the oxalomalate-
`administered mice than the control mice (Fig. 4A). These
`data indicate that GSSG in the kidney mitochondria of the
`oxalomalate-administered mice was not reduced as efficiently
`as in that of the control mice. NADPH, required for GSH
`generation by glutathione reductase, is an essential factor for
`the cellular defense against oxidative damage. The ratio for
`[NADPH]/[NADP+ + NADPH]
`(NADPt) was
`mitochondrial
`significantly decreased in mice treated with 2 Gy of
`γ-irradiation; however, the decrease in this ratio was much
`more pronounced in the kidneys of the oxalomalate-adminis-
`tered mice (Fig. 4B). Alterations in mitochondrial integrity
`and function may play an important role in the apoptotic
`cascade. MPT, associated with the opening of large pores in
`the mitochondrial membranes, is a very important event in
`apoptosis, and ROS is one of the major stimuli that change
`MPT [23]. The kidneys of
`the oxalomalate-treated mice
`showed a significant change in Δφm determined with JC-1
`(Fig. 4C). To determine if changes in MPT were accompanied
`by changes in intracellular ROS, the levels of intracellular
`
`Fig. 2. Effects of oxalomalate on ionizing radiation-induced apoptosis in
`dissociated kidney epithelial cells from mice. (A) Activation of caspase-3 by 2
`Gy of γ-irradiation can be followed by flow cytometry. Dissociated kidney cells
`from mice were stained with anti-cleaved caspase-3 IgG and analyzed by flow
`cytometry (left). Percentage of apoptotic cells calculated from data obtained by
`flow cytometry. Each value represents the mean ± SD of samples from five
`animals. (Right) *p < 0.01 vs irradiated control mice. (B) Characteristic DNA
`fragmentation in the kidney cells from mice was determined by the TUNEL
`assay and examined by fluorescence microscopy. TUNEL-positive cells were
`shown by bright FITC staining of nuclei.
`
`caspase-3-positive cells had increased and was significantly
`higher in the kidneys of the oxalomalate-treated mice than in
`the control mice on exposure to a clinically relevant dose of 2
`Gy. The DNA fragmentation evaluated by the TUNEL assay
`was significantly increased in the kidney cells of
`the
`oxalomalate-treated mice compared to the control mice when
`the mice were exposed to 2 Gy of γ-irradiation (Fig. 2B).
`
`Cellular oxidative damage
`
`To investigate whether the difference in apoptotic cell death
`of kidney cells from the oxalomalate-treated or the control
`mice on exposure to ionizing radiation is associated with ROS
`formation, the levels of intracellular hydrogen peroxide in the
`kidneys were evaluated. As shown in Fig. 3A, a significantly
`higher intracellular level of H2O2 was observed in the kidneys
`of the oxalomalate-administered mice compared to the control
`mice with the exposure of 2 Gy of γ-irradiation. These data
`strengthen the conclusion that IDPm provided protection from
`the ionizing radiation-induced apoptosis by decreasing the
`steady-state level of
`intracellular oxidants. As indicative
`markers of oxidative damage to cells,
`the occurrences of
`oxidative DNA damage, protein oxidation, and lipid peroxida-
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`Fig. 3. Effects of oxalomalate on the cellular oxidative damage of the kidneys of mice exposed to ionizing radiation. (A) Production of hydrogen peroxide in the
`kidneys of mice exposed to ionizing radiation was determined by the method described under Experimental procedures. Each value represents the mean ± SD (n = 5).
`*p < 0.01 vs irradiated control mice. (B) Lipid peroxidation of the kidneys of mice after exposure to ionizing radiation. The level of MDA accumulated in the mice
`unexposed and exposed to γ-irradiation was determined by using a TBARS assay. Each value represents the mean ± SD (n = 4). *p < 0.01 vs irradiated control mice. (C)
`Protein carbonyl content of the kidneys of mice exposed to ionizing radiation. Protein carbonyls were measured in cell-free extracts with the use of DNPH. Each value
`represents the mean ± SD (n = 5). *p < 0.01 vs irradiated control mice. (D) 8-OH-dG levels in the kidneys of irradiated mice. 8-OH-dG levels reflected by the binding of
`avidin-TRITC were visualized fluorescence microscope.
`
`peroxides in the mitochondria of mouse kidney were evaluated
`by confocal microscopy with the oxidant-sensitive probe DHR
`123. As shown in Fig. 4D, the intensity of fluorescence was
`significantly higher
`in the kidneys of
`the oxalomalate-
`administered mice compared to that in the mitochondria of
`kidneys from the control mice when mice were exposed to 2
`Gy of γ-irradiation. These results indicate that
`ionizing
`radiation most likely leads to increased mitochondrial injury
`while IDPm protects mitochondria from oxidative damage.
`Mitochondrial injury is often followed by the depletion of
`intracellular ATP level. As shown in Fig. 4E, when mice were
`exposed to 2 Gy of γ-irradiation the ATP level was
`significantly decreased in the kidneys of the oxalomalate-
`administered mice compared to the control mice, suggesting a
`protective role of IDPm against the loss of intracellular ATP
`levels.
`
`Modulation of the apoptotic marker proteins
`
`We evaluated changes in the apoptotic marker proteins as a
`result of
`ionizing radiation and the influence of
`IDPm
`expression on these proteins. The role of mitochondrial
`pathways of apoptosis in the ionizing radiation-induced cell
`death of mice kidneys was examined by immunoblot analysis
`of the abundance of Bax, Noxa, or PUMA, a proapoptotic
`protein. As shown in Fig. 5, the amount of Bax, Noxa, or
`
`PUMA was increased after exposure to ionizing radiation, and
`it was significantly increased in the kidneys of the oxaloma-
`late-administered mice compared to the control mice. The
`release of cytochrome c from mitochondria, a critical event
`provoking a cascade of caspases and eventually irreversible
`cell death [24], was increased after exposure to ionizing
`radiation. A marked increase of cytochrome c release was
`observed in the kidneys of the oxalomalate-administered mice
`compared to control mice. Caspase-3 activation in the mouse
`kidney was assessed by immunoblot analysis of kidney lysates
`from mice that had been exposed to 2 Gy of γ-irradiation.
`Ionizing radiation induced cleavage of caspase-3; however, the
`cleavage was significantly increased by the reduced activity of
`IDPm.
`Ionizing radiation also induced the formation of
`fragments which represents proteolytic cleavage of PARP,
`indicating an oncoming apoptotic process. The cleaved
`products of PARP increased markedly in the kidneys of the
`oxalomalate-administered mice compared to the control mice
`on exposure to ionizing radiation. Taken together, ionizing
`radiation-induced cleavage of procaspase-3 into the active
`form of caspase-3 and caspase-3 induces degradation of PARP.
`Ionizing radiation induces double-strand breaks in cells. One
`of the key components in damaged DNA recognition and
`signaling after γ-irradiation is the tumor suppressor protein
`p53 [25]. p53 protein levels determined by Western blotting
`were increased as a result of ionizing radiation and the reduced
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`49
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`Fig. 4. Effects of oxalomalate on the mitochondrial redox status and the mitochondrial function of the kidneys of mice exposed to ionizing radiation. Ratio of GSSG
`versus the total GSH pool (A) and NADPH versus the total NADP pool (B) in kidney mitochondria from mice. Each value represents the mean ± SD (n = 5). *p < 0.01
`vs irradiated control mice. (C) Bivariate JC-1 analysis of mitochondrial membrane potential in the dissociated kidney cells from mice by flow cytometry. (D) Effect of
`oxalomalate on mitochondrial ROS generation. DHR 123 was employed to detect mitochondrial ROS. Fluorescence was measured by flow cytometry. (E) Effect of
`oxalomalate on the levels of intracellular ATP. The kidneys of mice were assayed for intracellular ATP content. Each value represents the mean ± SD (n = 5). *p < 0.01
`vs irradiated control mice.
`
`activity of IDPm by oxalomalate enhanced the radiation-
`induced up-regulation of p53 levels. p21, as a regulator protein
`and a p53 target gene, modulates the kinase activities of a
`variety of cyclin-dependent kinases [26]. The p21 induction
`was apparent in the mouse kidney after γ-irradiation, and the
`
`Fig. 5. Immunoblot analysis of apoptotic marker proteins in the kidney cells
`from mice exposed to ionizing radiation. Cell extracts were subjected to 10-
`12.5% SDS-PAGE and immunoblotted with antibodies against p53, Bax,
`PUMA, Noxa, cytochrome c, cleaved caspase-3, cleaved PARP, or p21. β-Actin
`was run as an internal control.
`
`level of induction was much higher in the kidneys of the
`oxalomalate-administered mice.
`
`Discussion
`
`Ionizing radiation is toxic to living cells and organisms
`because it induces deleterious structural changes in essential
`biomolecules. A significant part of the initial damage done to
`U
`OH, which
`cells by ionizing radiation is due to formation of
`reacts with almost all cellular components to induce oxidative
`damage to DNA,
`lipid peroxidation, and protein oxidation
`[3,27,28]. DNA is a particularly important target, suffering
`double- and single-strand breaks, deoxyribose damage, and
`base modification. Of the total damage to DNA caused by
`ionizing radiation, as much as 80% may result from radiation-
`induced water-derived free radicals and secondary carbon-based
`radicals [1]. In addition to the generation of hydroxyl radicals,
`the hydrated electrons formed by ionizing radiation can reduce
`
`
`it to O2–. O2– can dismutate to H2O2 with the possibility of extra
`U
`OH production by a metal-catalyzed Fenton reaction [2]. Free
`radicals can also initiate a variety of cellular signal transduction
`pathways that may either aid the cell in coping with the excess
`oxidative stress resulting from radiation or set into motion
`pathways that lead to the destruction of cells damaged beyond
`the repair capabilities of the cell [29,30]. Therefore, there are
`several reports to suggest that antioxidant enzymes may prevent
`oxidative damage and cell death including apoptosis caused by
`ionizing radiation.
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`IDPm is a key enzyme in cellular defense against oxidative
`damage by supplying NADPH in the mitochondria. NADPH is
`an essential cofactor for the regeneration of GSH, the most
`abundant
`low-molecular-mass thiol
`in most organisms, by
`glutathione reductase in addition to its critical role for the
`activity of the NADPH-dependent thioredoxin system [9,10].
`Mitochondrial GSH becomes critically important against ROS-
`mediated damage because it not only functions as a potent
`antioxidant but is also required for the activities of mitochon-
`drial glutathione peroxidase and mitochondrial phospholipid
`hydroperoxide glutathione peroxidase [31], which removes
`mitochondrial peroxides. The oxidized form of thioredoxin,
`with a disulfide bridge between the half-cystines, can be
`reduced by NADPH in the presence of a flavoprotein,
`thioredoxin reductase [32]. Reduced thioredoxin may provide
`reducing equivalents to mitochondrial thioredoxin peroxidase
`family/peroxiredoxin family including peroxiredoxin III/protein
`SP-22 [33–35] and peroxiredoxin V/AOEB166 [36]. Therefore,
`any mitochondrial NADPH producer,
`if present, becomes
`critically important for cellular defense against ROS-mediated
`damage. Suppression of mitochondrial NADPH and GSH levels
`by reduction of IDPm activity by oxalomalate in turn enhanced
`the oxidative stress and concomitant ROS-mediated cell death.
`Therefore, it was plausible to assume that IDPm may play a role
`in preventing apoptosis caused by ionizing radiation in cells.
`The aim of the present work was to evaluate the role of IDPm in
`protecting mice from ionizing radiation in regard to apoptotic
`cell death, cellular redox status, mitochondrial dysfunction, and
`oxidative damage to cells. In the present study, a temporal
`pattern of events reflecting ionizing radiation-induced apoptosis
`was observed, starting from the elevation of ROS level,
`followed by MPT alteration, caspase-3 activation, and DNA
`fragmentation. The reduction of IDPm activity by oxalomalate
`significantly increased ROS level and enhanced the whole
`apoptotic pathway.
`The involvement of mitochondria in apoptosis has been
`extensively discussed [37]. All the changes caused by ionizing
`radiation are compatible with mitochondrial failure, encom-
`passing reduced production of ATP, generation of ROS, and
`modulation of mitochondrial membrane potential. A clear
`enhancement of such damages in the oxalomalate-treated mice
`compared to the control mice suggests that IDPm prevents a
`deterioration of the bioenergetic state.
`Cleavage of caspase-3 and its target protein such as PARP, a
`signature event of apoptosis, was induced by ionizing radiation.
`The reduction of IDPm activity by oxalomalate enhanced
`programmed cell death by increasing apoptotic features
`including caspase activation, and increasing proapoptotic
`molecules such as Bax, Noxa, and PUMA, presumably via
`preservation of redox status. Wild-type p53 tumor suppressor
`protein has been shown to be functionally necessary for growth
`inhibition and apoptosis following exposure to ionizing
`radiation [38]. Levels of p53 were increased in mouse kidney
`after radiation treatment. The reduction of IDPm activity
`enhanced the radiation-induced increase in p53 levels, which
`correlates with an enhancement
`in radiation-induced DNA
`damage.
`
`ionizing radiation-induced apoptosis by
`Regulation of
`oxalomalate will likely have significance in cancer treatment.
`Cancer cells increasingly become resistant
`to consecutive
`administration of chemotherapeutic drugs and radiation. There-
`fore, enhancement of tumor radioresponse may improve the
`therapeutic ratio of radiotherapy. This study indicates that
`oxalomalate enhances the susceptibility of ionizing radiation-
`induced apoptosis through inhibiting IDPm activity. The
`ovserved effects of oxalomalate in mice supported here offer
`the possibility of developing a modifier of radiation therapy.
`In conclusion, the present study demonstrates that IDPm
`abrogates the ionizing radiation-induced early production of
`ROS, leading to protection against apoptotic cell death. In this
`regard, oxalomalate, an inhibitor of IDPm, could play a role as a
`rediosensitizer in tumor radiotherapy.
`
`Acknowledgments
`
`This work was supported by grants of the National R and
`D Program for Cancer Control, Ministry of Health and
`Welfare, Republic of Korea (0420150-1), and a Nuclear
`Research Program from the Korea Science and Engineering
`Foundation (M2-0513-000102-05-B09-00-102-10).
`
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