[CANCER RESEARCH 63, 6885– 6893, October 15, 2003]
Protective Role of ␣-Phenyl-N-t-butylnitrone against Ionizing Radiation in
U937 Cells and Mice1
Jin Hyup Lee and Jeen-Woo Park2
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea
ABSTRACT
Ionizing radiation (IR) induces the production of reactive oxygen species (ROS), which play an important causative role in radiation damage.
␣-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spintrapping compounds for investigating the existence of free radicals in
biological systems. We investigated the protective role of PBN against IR
in U937 cells and mice. On exposure to IR, there was a distinct difference
between the control cells and the cells pretreated with PBN in regard to
viability, cellular redox status, and oxidative damage to cells. Lipid peroxidation, oxidative DNA damage, and protein oxidation were significantly lower in the cells treated with PBN when the cells were exposed to
IR. Although the activities of antioxidant enzymes were comparable in
PBN-treated and control cells, the [GSSG]:[GSH ⴙ GSSG] ratio and the
generation of intracellular ROS were higher and the [NADPH]:
[NADPⴙ ⴙ NADPH] ratio was lower in control cells compared with
PBN-treated cells. The IR-induced mitochondrial damage reflected by the
altered mitochondrial permeability transition, the increase in the accumulation of ROS, the reduction of ATP production, and the morphological change were significantly higher in control cells compared with
PBN-treated cells. PBN administration for 14 days with a daily dosage of
30 mg/kg provided substantial protection against killing and oxidative
damage to mice exposed to whole body irradiation. These data indicate
that PBN may have great application potential as a new class of in vivo,
nonsulfur-containing radiation protector.
INTRODUCTION
The damaging effect of IR on living cells is predominantly attributable to ROS3, including O2⫺, 䡠OH, and H2O2, generated by the
decomposition of water. The secondary radicals formed by the interaction of 䡠OH with organic molecules may also be of importance (1,
2). These oxygen-free radicals have the potential to damage critical
cellular components such as DNA, proteins, and lipids and eventually
results in physical and chemical damage to tissues that may lead to
cell death or neoplastic transformation (3).
The search for agents that protect against IR is important to those
at risk by virtue of environmental exposure or health-related treatment
and scientific study of the mechanism of radiation injury and cytotoxicity (4). Although no radioprotective drug available today has all
of the requisite qualities to be an ideal radioprotector, sulfhydryl
radioprotectors such as cysteine, cysteamine, cystamine, aminoethyliReceived 6/5/03; revised 7/22/03; accepted 7/30/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by a Nuclear Research Program from the Ministry of Science and Technology, Korea.
2
To whom requests for reprints should be addressed, at Department of Biochemistry,
College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
Phone: 82-53-950-6352; Fax: 82-53-943-2762; E-mail: [email protected].
3
The abbreviations used are: ROS, reactive oxygen species; IR, ionizing radiation;
PBN, ␣-phenyl-N-t-butylnitrone; DNPH, 2,4-dinitophenylhydrazine; DTNB, 5,5⬘-dithiobis(2-mitrobenzoic acid); TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; HNE, 4-hydroxynonenal; DNP, dinitrophenyl; DCFH-DA,
2⬘,7⬘-dichloro-fluoroscin diacetate; CMAC, t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin; DPPP, diphenyl-L-pyrenylphosphine; DHR 123, dihydrorhodamine
123; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMS, phenazine methosulfate; G6PDH, glucose 6-phosphate dehydrogenase; DCF, 2⬘,7⬘-dichlorofluorescein; FACS, fluorescence-activated cell
sorter; MDA, malondialdehyde; 8-OH-dG, 8-hydroxy-2⬘-deoxyguanosine; MTP, mitochondrial permeability transition; GSSG, oxidized glutathione; GSH, reduced glutathione.
sothiourea dihydrobromide, and mercaptoethyl guanidine are the best
radioprotectors known today. However, their use encounters two great
difficulties: their toxicity and the short period during which they are
active (5).
PBN is one of the most widely used spin-trapping compounds for
investigating the existence of free radicals in biological systems. PBN
reverses the age-related oxidative changes in the brains of old gerbils
(6), delays senescence in senescence-accelerated mice and normal
mice (7, 8), and it alleviates oxidative damage from ischemia/reperfusion injury (9). This phenomenon was accounted for by the fact that
PBN protected biologically important molecules from oxidative damage by efficiently trapping ROS, including O2⫺ (10). From a study
concerning the tissue distribution, excretion, and metabolism of PBN,
it was shown that this is rapidly absorbed, widely distributed inside
the body, and remains for a long period in many tissues when injected
i.p. into rats (11)
In the present study, the role of PBN in the cellular and in vivo
defense against IR was investigated using the U937 cells and mice.
There is mounting evidence that human monocytic U937 cells are
highly susceptible to many types of stresses. They also have a variety
of functions against external stresses. PBN-treated and untreated
U937 cells were expected to exhibit differences in sensitivity to the
toxic effects of IR. To determine whether such differences exist
between cells treated and untreated with PBN, viability, cellular redox
status, oxidative damage to cells, and mitochondrial damage were
examined on their exposure to IR. PBN was also administered to
mice, and in vivo radioprotective effect was assessed. This study
indicates that PBN may play an important role in regulating the
damage induced by IR, and PBN may have great application potential
as a new class of in vivo, nonsulfur-containing radiation protector.
MATERIALS AND METHODS
Materials. Hydrogen peroxide, PBN, pyrogallol, glutathione reductase,
G6PDH, tert-butyl hydroperoxide, ␤-NADP⫹, ␤-NADPH, GSSG, DNPH,
DTNB, xylenol orange, antirabbit IgG TRITC-conjugated secondary antibody,
and antirabbit IgG FITC-conjugated secondary antibody were obtained from
Sigma Chemical Co. (St. Louis, MO). Antihuman HNE-Michael adduct antibody and antihuman DNP antibody were obtained from Calbiochem (La Jolla,
CA). DCFH-DA, CMAC, DPPP, DHR 123, and the LIVE/DEAD viability/
cytotoxicity kit were purchased from Molecular Probes (Eugene, OR).
Cell Culture. Human premonocytic U937 cells (American Type Culture
Collection, Manassas, VA) were grown in RPMI 1640 culture supplemented
with 10% (v/v) fetal bovine serum, penicillin (50 units/ml), and 50 ␮g/ml
streptomycin at 37°C in a 5% CO2/95% air humidified incubator.
Mice. Adult male ICR mice were supplied through the production. The
animals were housed five per cage in a climate-controlled, circadian rhythmadjusted room and allowed food and water ad libitum. The animals were, on
average, 50 –70 days old and weighed between 20 and 30 g at the time of
irradiation. Experiments on mice were conducted according to the principles
outlined in the Guide for the Care and Use of Laboratory Animal prepared by
the Institute of Laboratory Animal Resources, National Research Council
(Washington, DC).
Irradiation and Cytotoxicity Assays. U937 cells were first grown on a
96-well plate at a density of 2 ⫻ 105 cells/well, until 80% confluence before
IR, and cell viability after IR was assessed by a novel tetrazolium compound,
MTS, and an electron coupling reagent, PMS. After culture for confluence
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optimization, various concentrations of PBN were applied to the cells and cells
were incubated for an additional 2 h at 37°C. PBN was prepared in 0.1%
ethanol and then diluted 100-fold in complete media. To control for 0.1%
ethanol in the pretreatment, a control group of cells was incubated in fresh
complete media with 1:100 vol of 0.1% ethanol for 2 h. After incubation, cells
were irradiated at room temperature with 137Cs source at a dose rate of 1
Gy/min. After 48 h of irradiation to cells, 20 ␮l of MTS/PMS solution were
added and incubated for another 4 h at 37°C in a humidified, 5% CO2
atmosphere. The conversion of MTS into aqueous, soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells.
The absorbance was read in an ELISA plate reader at 490 nm with a 620 nm
reference. Cell viability is expressed as a percentage of the absorbance seen in
the untreated control cells. Cell viability was also observed using a fluorescent
LIVE/DEAD viability assay, following the manufacturer’s protocol. Cells
were double-stained with calcein-AM and ethidium homodimer-1 and observed with a fluorescence microscope.
Toxicology of PBN and Whole Body Irradiation. To determine its maximal tolerated dose, solutions of PBN were freshly prepared in 0.9% NaCl.
Two groups of 15 mice each received either PBN or 0.9% NaCl. PBN was
administered before irradiation at a dose of 30 mg/kg in volumes equivalent to
1% of each animal’s weight to once daily for 2 weeks. Control mice were given
0.9% NaCl, and all injections were administered i.p. Survival was assessed up
to 30 days after injection without irradiation. To determine survival after whole
body irradiation, the same protocol for the PBN administration was applied,
and then the groups of 15 mice were transferred to round Plexiglas containers
(30.5 cm in diameter and 10.5 cm in height) with holes for ventilation. After
irradiation with a 137Cs source at a dose rate of 1 Gy/min, the mice were
returned to climate-controlled cages for observation. Survival was assessed 30
days after irradiation.
Preparation of Tissue Extracts. The tissue portions were homogenized
with a solution of 0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA (pH 7.2)
containing 8.5 ␮M leupeptin and 100 ␮g/ml aprotinin, using a homogenizer at
maximum speed for 15 s. Each sample was then centrifuged at 4000 ⫻ g for
15 min at 4°C, and the resulting supernatants were stored at ⫺20°C and used
for the assays. The protein concentration in the supernatant was measured by
the Bradford method using the Bio-Rad Protein Assay kit.
Enzyme Assay. Cells were collected at 1,000 ⫻ g for 10 min at 4°C and
were washed once with cold PBS. Cells were homogenized with a Dounce
homogenizer in sucrose buffer [0.32 M sucrose and 10 mM Tris-Cl (pH 7.4)].
Cell homogenates were centrifuged at 1,000 ⫻ g for 5 min, and the supernatants were centrifuged further at 15,000 ⫻ g for 30 min. The supernatants were
used to measure the activities of several cytosolic enzymes. Catalase activity
was measured with the decomposition of hydrogen peroxide, which was
determined by the decrease in absorbance at 240 nm (12). Superoxide dismutase activity in cell extracts was assayed spectrophotometrically using a
pyrogallol assay (13), in which one unit of activity is defined as the quantity
of enzyme that reduces the superoxide-dependent color change by 50%.
G6PDH activity was measured by following the rate of NADP⫹ reduction at
340 nm using the procedure described (14). Glutathione reductase activity was
quantified by the GSSG-dependent loss of NADPH (15) as measured at 340
nm. Glutathione peroxidase activity in the crude extracts was measured by the
standard indirect method based on NADPH oxidation by tert-butyl hydroperoxide in the presence of excess glutathione and glutathione reductase, as
described previously (16).
NADPH and GSH Levels. 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
(⑀ ⫽ 1.36 ⫻ 104 M⫺1cm⫺1), as described by Akerboom and Sies (18), and
GSSG was measured by the DTNB-GSSG reductase recycling assay after
treating GSH with 2-vinylpyridine (19). The total GSH level was measured in
0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.2 mg of
NADPH, 30 ␮g of DTNB, and 0.12 unit of glutathione reductase. The GSSG
level was measured by the same method as the total GSH level, but after
treatment of 1 ␮l of 2-vinylpyridine and 3 ␮l of triethanolamine for 1 h. The
intracelluar GSH level was also determined by using a GSH-sensitive fluorescence dye, CMAC. U937 cells (1 ⫻ 106 cells/ml) were incubated with 5 ␮M
CMAC cell tracker for 30 min. For in vivo visualization of GSH levels in
tissues, tissues were perfused with 5 ␮M CMAC for 60 min and the cryosec-
tions of tissues were prepared. The images of CMAC cell tracker fluorescence
by GSH was analyzed by the Zeiss Axiovert 200 inverted microscope at
fluorescence 4⬘,6-diamidino-2-phenylindole region (excitation, 351 nm; emission, 380 nm; Ref. 20).
Measurement of Intracellular ROS. Intracellular ROS production was
measured using the oxidant-sensitive fluorescent probe DCFH-DA with confocal microscopy (21). Cells were grown at 2 ⫻ 106 cells per 100-mm plate
containing slide glass coated with poly-L-lysine and maintained in the growth
medium for 24 h. Cells were treated with 10 ␮M DCFH-DA for 15 min and
exposed to 20 Gy of ␥-irradiation. Cells on the slide glass were washed with
PBS, and a cover glass was put on the slide glass. DCF fluorescence (excitation, 488 nm; emission, 520 nm) was imaged on a laser confocal scanning
microscope (DM/R-TCS; Leica) coupled to a microscope (Leitz DM REB).
For FACS analyses, measurement of DCF fluorescence in cells was made at
least 10,000 events/test using a FACS caliber flow cytometer (Becton Dickinson) with a fluorescein isothiocynate filter. For in vivo visualization of ROS
generation in tissues, tissues were perfused with 5 ␮M DCFH-DA for 25 min.
The DCF fluorescence from cryosections of tissues was observed with a
fluorescence microscope. Hydrogen peroxide oxidizes ferrous (Fe2⫹) to ferric
ion (Fe3⫹) selectively in dilute acid, and the resulting ferric ions can be
determined using a ferric-sensitive dye, xylenol orange, as an indirect measure
of intracellular hydrogen peroxide concentration. Cell-free extracts were added
ferrous oxidation in xylenol orange solution (0.1 mM xylenol orange, 0.25 mM
ammonium ferrous sulfate, 100 mM sorbitol, and 25 mM H2SO4) and incubated
in room temperature for 30 min, and absorbance was measured at 560 nm.
Hydrogen peroxide was used to draw standard curves as described (22).
Protein Carbonyl Content. The protein carbonyl content was determined
spectrophotometrically using the DNPH-labeling procedure as described (23).
The crude extract (⬃1 mg protein) was incubated with 0.4 ml of 0.2% DNPH
in 2 M HCl for 1 h at 37°C. The protein hydrazone derivatives were sequentially extracted with 10% (w/v) trichloroacetic acid, treated with ethanol/ethyl
acetate (1:1, v/v), and reextracted with 10% trichloroacetic acid. The resulting
precipitate was dissolved in 6 M guanidine hydrochloride, and the difference
spectrum of the sample treated with DNPH in HCl was examined versus a
sample treated with HCl alone. Results are expressed as nanomoles of DNPH
incorporated per milligram of protein calculated from an absorbtivity of 21.0
mM⫺1cm⫺1 at 360 nm for aliphatic hydrazones. The protein carbonyl contents
in U937 cells were also determined with DNP-specific antibody (1:200 dilution) and antihuman IgG FITC (excitation, 488 nm; emission, 520 nm) conjugate (1:800 dilution) as a secondary antibody, and then fluorescence was
observed using a fluorescence microscope.
Lipid Peroxidation. The lipid peroxidation in U937 cells was determined
with rabbit polyclonal anti-HNE-Michael adduct antibody (1:200 dilution) and
antihuman IgG TRITC conjugate (1:800 dilution). Lipid peroxidation was also
estimated by using a fluorescent probe, DPPP, as described by Okimoto et al.
(24). After U937 cells (1 ⫻ 106 cells/ml) were incubated with 5 ␮M DPPP for
15 min in the dark, cells were exposed to ionizing radiation. For in vivo
visualization of lipid peroxidation in tissues, tissues were perfused with 5 ␮M
DPPP for 30 min and the cryosections of tissues were prepared. The images of
DPPP fluorescence by reactive species were analyzed by the Zeiss Axiovert
200 inverted microscope at fluorescence 4⬘,6-diamidino-2-phenylindole region
(excitation, 351 nm; emission, 380 nm).
DNA Damage Analysis. 8-OH-dG levels of U937 cells were estimated by
using a fluorescent binding assay, as described by Struthers et al. (25). After
U937 cells were exposed to IR, cells were fixed and permeabilized with
ice-cold methanol for 15 min. DNA damage was visualized with avidinconjugated TRITC (1:200 dilution) for fluorescent microscope with a 540-nm
excitation and 588-nm emission.
Immunohistochemistry. Tissues from saline or PBN-administered mice
(n ⫽ 10, each) after irradiation were fixed by retrograde perfusion via the aorta
with 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). For paraffin
sections, tissue blocks containing all kidney, liver, and lung zones were
dehydrated and embedded in paraffin. The sections were dewaxed and rehydrated. For immunofluorescence labeling, the staining was performed using
fluorescence tag-conjugated secondary antibodies. To reveal antigens, sections
were put in a 1-mM TRIS solution (pH 9.0) supplemented with 0.5 mM EGTA
and heated in a microwave oven for 10 min. Nonspecific immunoglobulin
binding was prevented by incubating the sections in 50 mM NH4Cl for 30 min,
followed by blocking with PBS supplemented with 1% BSA, 0.05% saponin,
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PBN AS A RADIOPROTECTOR
and 0.2% gelatin. Sections were incubated overnight at 4°C with antihuman were calculated from spectrophotometric absorbance at 259 nm using an
HNE-Michael adducts antibody for lipid peroxidation, antihuman DNP adduct extinction coefficient of 1.54 ⫻ 104 M⫺1cm⫺1.
Statistical Analysis. The difference between two mean values was anaantibody for protein oxidation, and avidin-TRITC conjugated for DNA base
modification diluted in PBS supplemented with 0.1% BSA and 0.3% Triton lyzed by the Student’s t test and was considered to be statistically significant
X-100 (1:200 dilution). After rinsing with PBS supplemented with 0.1% BSA, when P ⬍ 0.05.
Replicates. Unless otherwise indicated, each result described here is rep0.05% saponin, and 0.2% gelatin, labeling was visualized with the fluorescence tag-conjugated antirabbit IgG TRITC conjugate (1:200 dilution) as a resentative of at least three separate experiments.
secondary antibody for antihuman HNE-Michael adduct antibody and antirabbit IgG FITC conjugate (1:200 dilution) as a secondary antibody for anti-DNP RESULTS
antibody diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100.
Microscopy was performed using a Zeiss fluorescence microscope.
Cell Killing. As shown in Fig. 1, when cultured U937 cells were
Cryosection Preparation. Adult mice were anesthetized with i.p. injec- exposed to ␥-irradiation, a dose-dependent decrease in cell viability
tions of pentobarbital sodium (1 ml/kg body weight). Tissues were quickly was observed. However, the cells pretreated with 2 mM PBN for 2 h
dissected from mice. Tissues were rinsed briefly in cold PBS three times before were significantly more resistant than control cells untreated with
freezing onto metal chucks for cryosectioning (26). Tissues were embedded in PBN. The protective effect of PBN was concentration dependent, and
Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC). After
PBN itself up to 10 mM was without effect on the viability of U937
snap-freezing, a block was obtained without changing the shape of the sample.
cells (data not shown). The protective effect of PBN against IR was
The block was then transferred into a cryostat chamber. A precooled adhesive
also confirmed by dual staining with calcein-AM and ethidium hotape (Instrumedics, Hackensack, NJ) was used to support a 5-␮m-thick section
as the block was being cut. The still frozen section, which adhered to the tape, modimer-1. IR caused lethal injury to U937 cells, and their nuclei
was then laminated to the cold adhesive-coated slide (Instrumedics). An UV were mostly stained with ethidium homodimer-1 to exhibit a red
flash was used to polymerize the adhesive coating into a hard solvent-resistant fluorescence. PBN (2 mM) pretreatment for 2 h decreased the proporplastic to tightly anchor the section to the slide. Finally, the tape was removed tion of red fluorescent nuclei of dying cells in ␥-irradiated cultures
and the slide was immersed in cold acetone (⫺20 to ⫺25°C), in which the ice (data not shown).
was dissolved but not melted (freeze substitution). The slide was then transCellular Redox Status. To investigate whether the difference in
ferred to 10% formaldehyde/0.25% glutaraldehyde/75% alcohol for 10 min at viability of U937 cells after exposure to IR is associated with ROS
room temperature.
formation, the levels of intracellular peroxides in the U937 cells were
MPT. Mitochondrial membrane potential was measured by the incorpora- evaluated by confocal microscopy with the oxidant-sensitive probe
tion of rhodamine 123 dye into the mitochondria, as described previously (27). DCFH-DA. As shown in Fig. 2A, an increase in DCF fluorescence
Cells (1 ⫻ 106) grown on poly-L-lysine-coated slide glasses were exposed to
was observed in U937 cells when they were exposed to 20 Gy of
IR. Cells were then treated with 5 ␮M rhodamine 123 for 15 min and excited
␥-irradiation. The increase in fluorescence was significantly reduced
at 488 nm with an argon laser. Cells were double-stained with 100 nM
MitoTracker Red, which is a morphological marker of mitochondria. The in cells pretreated with 2 mM PBN for 2 h. Similar results were also
fluorescence images at 520 nm were simultaneously obtained with a laser observed by FACS analyses (Fig. 2B). We also demonstrated the level
of intracellular H2O2 in cells irradiated in the presence and absence of
confocal scanning microscope.
Mitochondrial ROS. Cells were first grown on an easy flask 75 FILT at a PBN. The pretreatment of PBN resulted in a significantly lower
density of 1 ⫻ 106 cells/well, until 80% confluence before IR. After culture for intracellular level of H2O2 as compared with that of untreated control
confluence optimization, 2 mM PBN were applied to the cells, which were then with the exposure of 20 Gy of irradiation (Fig. 2C). These data
incubated for an additional 2 h at 37°C. After additional incubation, DHR 123 strengthen the conclusion that PBN provided protection from the
was used to detect mitochondrial ROS. U937 cells in PBS were incubated for cytotoxic actions of ␥-irradiation by decreasing the steady-state level
20 min at 37°C with 5 ␮M DHR 123 and cells were double-stained with 100 of intracellular oxidants.
nM MitoTracker Red. Cells were washed and resuspended in complete growth
GSH is one of the most abundant intracellular antioxidants, and
media, and IR was applied to the cells. The cells were then incubated for an
determination of changes in its concentration provides an alternative
additional 40 min. DHR 123 and Mitracker Red fluorescence were visualized
method of monitoring oxidative stress within cells. It has been shown
by a fluorescence microscope.
the GSH-sensitive fluorescent dye CMAC can be used as a useful
Transmission Electron Microscopy. Cells grown to 80% confluence were
either treated or untreated with PBN and exposed to IR, rinsed twice with PBS probe to evaluate the level of intracellular GSH (20). Cellular GSH
(pH 7.3), and centrifuged at 50 ⫻ g for 5 min. Cell pellets were immediately
fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer for 2 h at 4°C.
Cells were postfixed in osmium tetroxide (1%) for 30 min, washed with water,
and then subjected to a dehydration procedure using graded ethanol series. For
preparing the specimen, cells were embedded in Epon812 (Electron Microscopy Sciences, Fort Washington, PA), and two random areas were cut and
processed. The sections (60 –70 nm) were cut with an ultramicrotome (Soya
MT-7000), transferred to copper grids, and stained with uranyl acetate and lead
citrate. At least 40 cells of each sample were observed and photographed using
Hitachi H-7100 transmission electron microscope (Hitachi Co., Hitach, Japan)
at 75 kV.
Measurement of ATP Level. Intracellular ATP levels were determined by
using luciferin-luciferase (28). Cells (5 ⫻ 106) were collected by centrifugation, resuspended in 250 ␮l of extraction solution [10 mM KH2PO4 and 4 mM
MgSO4 (pH 7.4)], heated at 98°C for 4 min, and placed on ice. For ATP
measurement, an aliquot of a 50-␮l sample was added to 100 ␮l of reaction
solution [50 mM NaASO2 and 20 mM MgSO4 (pH 7.4)] containing 800 ␮g
Fig. 1. Resistance of control (f) and PBN-treated (Œ) U937 cells against IR. Cells
of luciferin-luciferase (Sigma Chemical Co.). Light emission was quantitated
5
in a Turner Designs TD 20/20 luminometer (Stratec Biomedical Systems, (2 ⫻ 10 /well) were grown in 96-well plates and then exposed to different doses of
␥-irradiation before measurement of cell viability by MTS assay. Cell viability is exBirkenfeld, Germany). For all experiments, ATP standard curves were run and pressed as a percentage of absorbance seen in the untreated control cells. Each value
were linear in the range of 5–2500 nM. Concentrations of ATP stock solution represents the mean ⫾ SD from five independent experiments.
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Fig. 2. Effects of PBN on peroxide level and
ROS generation. A, DCF fluorescence in U937
cells. Typical patterns of DCF fluorescence are
presented for U937 cells untreated and treated with
PBN before ␥-irradiation. Fluorescence images
were obtained under laser confocal microscopy
from three separate experiments. B, effect of PBN
on ␥-irradiation-induced ROS production. Cells
were exposed to ␥-irradiation and subjected to
FACS analyses. C, production of hydrogen peroxide in U937 cells was determined by the method
described in “Material and Methods.” Each value
represents the mean ⫾ SD from five independent
experiments.
levels in U937 cells treated with 20 Gy of ␥-irradiation were significantly decreased (Fig. 3A). However, the depletion of GSH was
significantly protected by the pretreatment of 2 mM PBN for 2 h. One
important parameter of GSH metabolism is the ratio of GSSG:total
GSH (GSHt), which may reflect the efficiency of GSH turnover.
When the cells were exposed to 20 Gy of ␥-irradiation, the ratio of
cellular [GSSG]:[GSHt] was significantly higher in control cells than
in PBN-treated cells (Fig. 3B). These data indicate that GSSG in
control cells was not reduced as efficiently as in PBN-treated cells.
These results suggest that decrease in the efficiency of GSH recycling
may be responsible for the higher concentration of intracellular per-
oxides. 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] was significantly decreased in cells treated with 20 Gy of ␥-irradiation, however,
the decrease in this ratio was much less pronounced in PBN-treated
cells (Fig. 3C). Despite their role in the cellular defense mechanism,
the antioxidant enzymes are susceptible to inactivation by ROS.
Previous studies have demonstrated that oxidative processes result in
the loss of key antioxidant enzymes, which may exacerbate oxidative
stress-mediated cytotoxicity. Whether the presence of PBN induced
concomitant alterations in the activity of major antioxidant enzymes
Fig. 3. Effect of PBN on the cellular redox
status of U937 cells exposed to IR. A, effect of
PBN on GSH levels. The fluorescence image of
CMAC-loaded cells was obtained under microscopy. B, ratios of GSSG versus total GSH pool.
Each value represents the mean ⫾ SD from three
independent experiments. C, ratios of NADPH versus NADP pool. Each value represents the
mean ⫾ SD from three independent experiments.
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Table 1 Effect of PBN on the antioxidant enzyme activities of U937 cells
Activitya
Antioxidant enzyme
Control
Control/PBN
Irradiated
Irradiated/PBN
SODb
Catalaseb
Glutathione
peroxidasec
Glutathione reductasec
G6PDHc
1.58 ⫾ 0.12
131 ⫾ 14
9.3 ⫾ 0.3
1.52 ⫾ 0.08
133 ⫾ 4.6
9.0 ⫾ 0.2
1.26 ⫾ 0.11
92.4 ⫾ 11
6.9 ⫾ 0.2
1.31 ⫾ 0.16
94.3 ⫾ 5.1
7.2 ⫾ 0.9
52.6 ⫾ 2.7
138 ⫾ 9.5
51.1 ⫾ 3.6
141 ⫾ 8.5
43.6 ⫾ 1.4
102 ⫾ 12
43.1 ⫾ 2.9
106 ⫾ 14
a
U937 cells were treated with 2 mM PBN for 2 h before 20 Gy of ␥-irradiation. Each
value represents the mean ⫾ SD from five independent experiments.
b
Enzyme activity represents units/mg protein.
c
Enzyme activity represents units/g protein.
was investigated. The activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, G6PDH, and glutathione reductase was decreased after exposure to ␥-irradiation, however, PBN did not significantly affect the activity of antioxidant
enzymes (Table 1). These results indicate that PBN may act as a direct
scavenger of ROS.
Cellular Damage. As indicative markers of oxidative damage to
cells, the occurrence of oxidative DNA damage, protein oxidation,
and lipid peroxidation were evaluated. To determine whether PBN
pretreatment decreased the sensitivity to protein damage, we performed carbonyl content measurements for protein oxidation after
cellular exposure to IR. Oxidative stress is known to introduce car-
bonyl groups into the amino acid side chains of proteins (23). Control
cells elicited an approximately 2-fold increase of carbonyl groups, as
compared with unirradiated cells when 20 Gy of IR were exposed.
Although the carbonyl content of PBN-treated cells also increased
with irradiation, the increase was significantly lower than that of
control cells (Fig. 4A). Protein oxidation was also visualized by
immunocytochemical method using anti-DNP antibody. As shown in
Fig. 4B, the fluorescent intensity that reflects the endogenous levels of
carbonyl groups in proteins was significantly increased when U937
cells were exposed to 20 Gy of ␥-irradiation, and the increase of
protein oxidation was markedly reduced in PBN-treated cells. The
increase in lipid peroxidation is proportional to the relative degree of
oxidative stress imposed on the cells. It is well established that
oxidative stress in various cells usually leads to accumulation of
potent, cytotoxic lipid peroxides such as MDA and HNE (29). Lipid
peroxidation was visualized by immunocytochemical method using
anti-HNE antibody. As shown in Fig. 4C, the fluorescent intensity that
reflects the endogenous levels of HNE adducts in proteins was significantly increased when U937 cells were exposed to 20 Gy of
␥-irradiation, and the increase of lipid peroxidation was markedly
reduced in PBN-treated cells. Recently, it has been shown that DPPP
is a suitable fluorescence probe to monitor lipid peroxidation within
cell membrane specifically. DPPP reacts with lipid hydroperoxides
stoichiometrically to give highly fluorescent product DPPP oxide
Fig. 4. Cell damage of U937 cells. A, protein carbonyl
content of U937 cells exposed to IR. Protein carbonyls
were measured in cell-free extracts using the method of
Levine et al. (23), with the use of DNPH. Each value
represents the mean ⫾ SD from five independent experiments. B, protein carbonyl content of U937 cells detected with DNP-specific antibody and antihuman IgG
FITC conjugate as a secondary antibody. C, lipid peroxidation in U937 cells was detected with polyclonal antiHNE-Michael adduct antibody and antihuman IgG
TRITC conjugate. D, visualization of lipid peroxidation
in U937 cells exposed to ␥-irradiation. Cells (1 ⫻ 106/
ml) were stained with 5 ␮M DPPP for 15 min. Fluorescence images were obtained under microscopy. E, 8-OHdG levels in U937 cells. The cells were fixed and
permeabilized immediately after exposure to ␥-irradiation. 8-OH-dG levels reflected by the binding of avidinTRITC were visualized by a fluorescence microscope.
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PBN AS A RADIOPROTECTOR
Fig. 5. Effects of PBN on mitochondrial structure and function. A, effect of PBN on MTP. MPT
of U937 cells was measured by the incorporation of
rhodamine 123 dye into the mitochondria. B, effect
of PBN on mitochondrial ROS generation. DHR
123 was used to detect mitochondrial ROS. DHR
123 fluorescenec was visualized by a fluorescence
microscope. C, effect of PBN on mitochondrial
ultrastructure. Cells untreated and treated with PBN
were exposed to ␥-irradiation, and their mitochondrial structures were then examined under transmission electron microscopy. D, effect of PBN on
the levels of intracellular ATP. Control and PBNtreated U937 cells were irradiated and assayed for
intracellular ATP content. Each value represents
the mean ⫾ SD from five independent experiments.
(25). DPPP fluorescent intensity was increased markedly in untreated
cells, whereas it was increased slightly in PBN-treated cells after
exposure to IR (Fig. 4D). 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. Because 8-OHdG causes misreplication of DNA (30), it has been implicated as a
possible cause of mutation and cancer. Therefore, 8-OH-dG has been
used as an indicator of oxidative DNA damage in vivo and in vitro
(31). Recently, it has been shown that the 8-OH-dG level is specifically measured by a fluorescent binding assay using avidin-conjugated TRITC (25). The fluorescent intensity that reflects the endogenous levels of 8-OH-dG in DNA was significantly increased in
untreated cells on exposure to IR. In contrast, the overall DNA
appeared to be markedly protected in PBN-treated cells, even after
exposure to the same dose of IR (Fig. 4E). These results indicate that
PBN seems to protect cells from oxidative damage caused by IR.
Mitochondrial Damage. MPT is a very important event in both
necrosis and apoptosis, and ROS is one of the major stimuli that
change MPT (32). To answer whether PBN modulates the MPT on
exposure to IR, we determined the change in MPT by intensity of
fluorescence emitting from a lipophilic cation dye, rhodamine 123.
Significantly less rhodamine 123 dye was taken up by the mitochondria of untreated cells, compared with PBN-treated cells (Fig. 5A).
The levels of intracellular peroxides in the mitochondria of U937 cells
were evaluated by confocal microscopy with the oxidant-sensitive
probe DHR 123. As shown in Fig. 5B, the intensity of fluorescence
was significantly lower in cells pretreated with 2 mM PBN for 1 h
when compared with that in the mitochondria of untreated cells when
U937 cells were exposed to 20 Gy of ␥-irradiation. MPT precedes the
cellular injury accompanied with significant changes in mitochondrial
structures (33). Therefore, we also examined the mitochondrial morphology of different U937 cells under electron microscopy (Fig. 5C).
Normal shapes of mitochondrial cristae in PBN-treated cells were
observed, whereas abnormal or substantially damaged mitochondrial
cristae were evident in untreated cells on exposure to IR. Mitochondria of untreated cells were extremely swollen and frequently lacked
typical cristae but contained their vesicular remnants and severely
collapsed membranes. These results indicate that IR most likely leads
to increased mitochondrial injury, whereas PBN protects mitochondria from oxidative damage. Mitochondrial injury is often followed by
the depletion of intracellular ATP level. As shown in Fig. 5D, when
U937 cells were exposed to 20 Gy of ␥-irradiation, the ATP level was
decreased only by 19% in PBN-treated cells, whereas it was reduced
by 51% in cells not treated with PBN, suggesting a protective role of
PBN against the loss of intracellular ATP levels.
In Vivo Radioprotection. Mice treated with PBN at 30 mg/kg
daily for 2 weeks survived with no apparent adverse effects after 30
days. ICR male mice received i.p. injections of 0.9% NaCl or PBN at
30 mg/kg daily for 14 days, and then they were subjected to 8 Gy of
whole body irradiation. Survival was monitored for 30 days, and the
results are shown in Fig. 6. PBN administration before irradiation
provided significant protection compared with control animals receiving 0.9% NaCl.
Mouse Redox Status. The livers from PBN-administered mice
before ␥-irradiation showed the lower level of hydrogen peroxide
(Fig. 7A), the lower level of DCF fluorescence (Fig. 7B), and suppressed depletion of the GSH level (Fig. 7C) compared with mice not
administered PBN. The ratio for [GSSG]:[GSHt] was lower (Fig. 7D)
and the ratio for [NADPH]:[NADPH ⫹ NADP⫹] was higher (Fig.
Fig. 6. PBN effect on the radiation sensitivity of ICR mice. Groups of 15 ICR mice
received an injection of either PBN (30 mg/kg, i.p.; Œ) or 0.9% NaCl (F) for 2 weeks,
were exposed to 8 Gy of whole body irradiation, and were then observed over 30 days for
survival.
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PBN AS A RADIOPROTECTOR
Fig. 7. Effects of PBN on redox status of the
livers from mice exposed to IR. A, production of
hydrogen peroxide in the livers from mice was
determined by the method described in “Material
and Methods.” Each value represents the
mean ⫾ SD from five independent experiments. B,
DCF fluorescence in the livers. Typical patterns of
DCF fluorescence are presented for livers from
mice untreated and treated with PBN before ␥irradiation. Fluorescence images were obtained under laser confocal microscopy from three separate
experiments. C, effect of PBN on GSH levels in the
livers from mice exposed to ␥-irradiation. The fluorescence image of CMAC-loaded cells was obtained under microscopy. D, ratios of GSSG versus
total GSH pool. Each value represents the
mean ⫾ SD from three independent experiments.
E, ratios of NADPH versus NADP pool. Each
value represents the mean ⫾ SD from three independent experiments.
7E) in the livers of PBN-administered mice before ␥-irradiation. The
kidneys and the lungs showed the similar results.
Tissue Damage. In the livers of untreated mice, carbonyl groups in
proteins were increased significantly after whole body irradiation.
Administration of PBN markedly prevented the increase in carbonyl
groups in proteins (Fig. 8A). The increase in DPPP fluorescence was
also inhibited by the treatment with PBN (Fig. 8B). Sections of liver
were immunohistochemically stained for HNE- and DNP-adduct using polyclonal HNE- and DNP-specific antibodies. The livers from
PBN-administered mice showed much less staining for both DNPand HNE-adducts (Figs. 8, C and D). Staining for these adducts was
similar in the sections of kidneys and lungs. As another marker of
oxidative damage in the liver, we measured the formation of 8-OH-dG
in the liver DNA. The result showed that the lower amount of
8-OH-dG reflected by the lower level of avidin-TRITC fluorescence
in PBN-administered mice than in non-PBN-administered mice (Fig.
8E). As shown in Fig. 8F, the livers from non-PBN-administered mice
showed the lower level of ATP compared with untreated mice, suggesting the role of PBN against the loss of mitochondrial function
caused by IR. The kidneys and the lungs showed the similar results.
DISCUSSION
IR 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 cells by IR caused by formation of 䡠OH,
which reacts with almost all cellular components to induce oxidative
damage to DNA, lipid peroxidation, and protein oxidation (3, 34, 35).
DNA is a particularly important target, suffering double- and singlestrand breaks, deoxyribose damage, and base modification (36). Of
the total damage to DNA caused by IR, as much as 80% may result
from radiation-induced water-derivative free radicals and secondary
carbon-based radicals (1). In addition to the generation of hydroxyl
radicals, the hydrated electrons formed by IR can reduce it to O2⫺.
O2⫺ can dismutate to H2O2 with the possibility of extra 䡠OH production by metal-catalyzed Fenton reaction (2). Therefore, there is considerable literature to suggest that free radical scavengers can be used
to prevent oxidative damage caused by IR. GSH, GSH precursors, and
thiol compounds have been used as radioprotectors. However, toxicities including the possible biological actions of sulfur-centered radicals produced by loss of H from thiols must not be ignored (36).
PBN, a lipophilic nitron compound, has been used widely as a spin
trap in vitro. PBN not only effectively scavenges ROS but also
suppresses the chain reactions leading to lipid peroxidation by trapping lipid radicals. PBN is not acutely toxic and, thus, has been used
in animal studies (11). There is mounting evidence that PBN has an
ameliorative effect on a variety of functions under acute oxidative
stress conditions (6, 9, 10). Therefore, it was plausible to assume that
PBN may play a role in preventing oxidative damage caused by IR in
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PBN AS A RADIOPROTECTOR
Fig. 8. Effects of PBN on oxidative damage of
the livers from mice exposed to IR. A, protein
carbonyl content after IR in mice received an injection of either PBN or 0.9% NaCl. Each value
represents the mean ⫾ SD from three independent
experiments. B, visualization of lipid peroxidation
in the livers from mice exposed to ␥-irradiation.
The livers were perfused with 5 ␮M DPPP for 30
min, and the cryosections of tissues were prepared.
Fluorescence images were obtained under microscopy. C, lipid peroxidation in the livers from irradiated mice was detected with polyclonal antiHNE-Michael adduct antibody and antihuman IgG
TRITC conjugate. D, protein carbonyl content of
the livers from irradiated mice detected with DNPspecific antibody and antihuman IgG FITC conjugate as a secondary antibody. E, 8-OH-dG levels in
the livers from irradiated mice. 8-OH-dG levels
reflected by the binding of avidin-TRITC were
visualized by a fluorescence microscope. F,
changes in the ATP content of the livers after
␥-irradiation. Control and PBN-treated mice were
irradiated and assayed for intracellular ATP content
from livers. Each value represents the mean ⫾ SD
from five independent experiments.
cells and animals. The aim of the present work was to evaluate the role
of PBN in protecting U937 cells and mice from IR in regard to cell
death, animal mortality, cellular redox status, and oxidative damage to
cells and tissues.
Biological systems have evolved to develop an effective and complicated network of defense mechanisms including antioxidant enzymes and small molecular weight antioxidants to cope with lethal
oxidative environments. The antioxidant enzymes were susceptible to
inactivation by ROS, however, the activity of antioxidant enzymes
were not significantly affected by PBN. GSH is known to play a role
in protecting cells against IR. Treatment with buthionine sulfoximine
to inhibit GSH synthesis increases radiosensitivity (37). The depletion
of intracellular GSH and the increase in the ratio of [GSSG]:[GSHt],
which reflects the efficiency of GSH turnover, were significantly
reduced by PBN. The ratio for [NADPH]:[NADP⫹ ⫹ NADPH], the
other parameter that reflects the cellular redox status and the avail-
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PBN AS A RADIOPROTECTOR
ability of the reducing equivalent for GSH turnover by glutathione
reductase, was significantly increased by PBN. These results indicate
that IR results in the perturbation of cellular redox balance presumably by depletion of GSH and NADPH pools and PBN may shift the
balance to antioxidant condition.
It is well established that mitochondrial dysfunction is directly and
indirectly involved in a variety of pathological states. All of the
changes caused by IR are compatible with mitochondrial failure,
encompassing reduced production of ATP, generation of ROS, accumulation of rhodamine 123 that reflect mitochondrial swelling or
changes in the mitochondrial inner membrane, and changes in mitochondrial morphology. A clear suppression of such damages indicates
that PBN prevents a deterioration of the bioenergetic state.
PBN is not only an in vitro but in vivo radioprotector. The radioprotective effect of PBN reflected by suppression of lethality was
evident 30 days after exposure to radiation. The measurement of lipid
peroxidation, protein oxidation, and oxidative DNA damage in livers,
kidneys, and lungs from mice exposed to ␥-irradiation indicates that
the damage caused by IR was similar in these tissues and that PBN
protects damage in tissues in a similar manner. To confirm oxidative
damage in irradiated mice because of the pro-oxidant status, the
formation of ROS in tissues was measured. PBN administration
showed the tendency to lower the formation of ROS. The observed
beneficial effects of PBN in mice supported here offer the possibility
of developing antioxidant approaches to treating damage caused by
IR. In conclusion, PBN is effective in protecting cells and mice from
oxidative stress caused by IR, and alleviated damage suggests that
further study of PBN or similar compounds is warranted.
ACKNOWLEDGMENTS
We are grateful to Drs. I. S. Kim and J. S. Suh (Kyungpook National
University) for help.
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Protective Role of α-Phenyl-N-t-butylnitrone against Ionizing
Radiation in U937 Cells and Mice
Jin Hyup Lee and Jeen-Woo Park
Cancer Res 2003;63:6885-6893.
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