Carcinogenesis vol.25 no.8 pp.1435--1442, 2004
doi:10.1093/carcin/bgh139
The use of N-t-butyl hydroxylamine for radioprotection in cultured cells and mice
Jin Hyup Lee1, In San Kim2 and Jeen-Woo Park1,3
1
2
Department of Biochemistry, College of Natural Sciences and College of
Medicine, Kyungpook National University, Taegu 702-701, Korea
3
To whom correspondence should be addressed
Email: [email protected]
Exposure of cells to ionizing radiation leads to formation of
reactive oxygen species (ROS) that are associated with
radiation-induced cytotoxicity. Therefore, compounds
that scavenge ROS may confer radioprotective effects.
Recently, it has been shown that the decomposition product
of the spin-trapping agent a-phenyl-N-t-butylnitrone
(PBN), N-t-butyl hydroxylamine (NtBHA), mimics PBN
and is much more potent in delaying ROS-associated senescence. We investigated the protective role of NtBHA
against ionizing radiation in U937 cells and mice. Viability
and cellular oxidative damage reflected by lipid peroxidation, oxidative DNA damage and protein oxidation were
significantly lower in the cells treated with NtBHA when
the cells were exposed to ionizing radiation. The modulation of cellular redox status was more pronounced in control cells compared with NtBHA-treated cells. The ionizing
radiation-induced mitochondrial damage reflected by the
altered mitochondrial permeability transition, the increase
in the accumulation of ROS and the reduction of ATP
production was significantly higher in control cells compared with NtBHA-treated cells. NtBHA administration
before irradiation at 5 mg/kg daily for 2 weeks provided
substantial protection against killing and oxidative damage
to mice exposed to whole-body irradiation. These data
indicate that NtBHA may have great application potential
as a new class of in vivo, non-sulfur containing radiation
protector.
Introduction
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
Abbreviations: CMAC, t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin; DCFH-DA, 20 ,70 -dichloro-fluoroscin diacetate; DHR, dihydrorhodamine; DNP, dinitrophenyl; DPPP, diphenyl-1-pyrenylphosphine; FACS,
fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; HNE,
4-hydroxynonenal; MBA, malondialdehyde; MPT, mitochondrial membrane
potential transition; NtBHA, N-t-butyl hydroxylamine; 8-OH-dG, 8-hydroxy20 -deoxyguanosine; PBN, a-phenyl-N-t-butylnitrone; ROS, reactive oxygen
species; TBARS, thiobarbituric acid-reactive substances; TRITC, tetramethylrhodamine isothiocyanate.
Carcinogenesis vol.25 no.8 # Oxford University Press 2004; all rights reserved.
the potential to damage critical cellular 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).
Since ROS appear to be mediators of the cellular damage
induced by ionizing radiation, compounds that regulate the
fate of such species may be of great importance in the protection of cells against radiation-induced damage. The search for
agents that protect against ionizing radiation is important to
those at risk by virtue of environmental exposure or healthrelated treatment and scientific study of the mechanism of
radiation injury and cytotoxicity (4). Although no radioprotective drug available today has all the requisite qualities to
be an ideal radioprotector, sulfhydryl radioprotectors such as
cysteine, N-acetylcysteine, cysteamine, cystamine, aminoethylisothiourea 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).
We recently reported that a-phenyl-N-t-butylnitrone (PBN)
protects damage caused by ionizing radiation in vitro and
in vivo (6). PBN is a spin-trapping agent that traps free radicals
and protects against damage in different models such as
inflammation, ischemia reperfusion and aging (7). The Ames
group recently reported that the decomposition product of
PBN, N-t-butyl hydroxylamine (NtBHA), mimics PBN and is
much more effective delaying senescence of IMR90 cells (8).
In this study, the role of NtBHA in the cellular and in vivo
defence against ionizing radiation was investigated using
U937 cells and mice. To determine differences in sensitivity
to the toxic effects of ionizing radiation between U937 cells
treated and untreated with NtBHA, viability, cellular redox
status, oxidative damage to cells and mitochondrial damage
were examined upon their exposure to ionizing radiation.
NtBHA was also administered to mice, and in vivo radioprotective effect was assessed. Our results demonstrate that
NtBHA may play an important role in regulating the damage
induced by ionizing radiation and NtBHA may have great
application potential as a new class of in vivo, non-sulfur
containing radiation protector.
Materials and methods
Materials
NtBHA, 2,4-dinitophenylhydrazine (DNPH), 5,50 -dithio-bis(2-nitrobenzoic
acid) (DTNB), pyrogallol, xylenol orange, anti-rabbit IgG tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary antibody and anti-rabbit
IgG fluorescein isothiocyanate (FITC) conjugated secondary antibody were
obtained from Sigma Chemical (St Louis, MO). Anti-human 4-hydroxynonenal (HNE)-Michael adduct antibody and anti-human dinitrophenyl (DNP)
antibody were obtained from Calbiochem (La Jolla, CA). 20 ,70 -Dichlorofluoroscin diacetate (DCFH-DA), t-butoxycarbonyl-Leu-Met-7-amino-4chloromethylcoumarin (CMAC), diphenyl-1-pyrenylphosphine (DPPP),
dihydrorhodamine (DHR) 123, and LIVE/DEAD viability/cytotoxicity kit
were purchased from Molecular Probes (Eugene, OR).
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J.H.Lee, I.S.Kim and J.-W.Park
Cell culture and cytotoxicity assay
Human premonocytic U937 cells (American Type Culture Collection,
Rockville, MD) were grown in RPMI 1640 culture medium supplemented
with 10% (v/v) FBS, penicillin (50 U/ml), and 50 mg/ml streptomycin at
37 C in a 5% CO2 /95% air humidified incubator. For cytotoxicity assay,
U937 cells were first grown on a 96 well plate at a density of 2 105 cells/
well. After culture for confluence optimization, various concentrations of
NtBHA were applied to the cells and cells were incubated for additional 2 h
at 37 C. After incubation, cells were irradiated at room temperature with 137 Cs
source at a dose rate of 1 Gy/min. After 48 h of irradiation to cells, 20 ml of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS)/phenazine methosulfate solution was added
and incubated for another 4 h at 37 C. The absorbance was read in an ELISA
plate reader at 490 nm with a 620 nm reference. 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.
anti-human 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. Thiobarbituric acid-reactive substances
(TBARS) were determined as an independent measurement of lipid peroxidation. Samples were evaluated for malondialdehyde (MDA) production using a
spectrophotometric assay for TBARS (16). The lipid peroxidation in U937
cells was determined with rabbit polyclonal anti-HNE Michael adduct antibody (1:200 dilution) and anti-human IgG TRITC conjugate (1:800 dilution).
Lipid peroxidation was also estimated by using a fluorescent probe DPPP as
described by Okimoto et al. (17). After U937 cells (1 106 cells/ml) were
incubated with 5 mM DPPP for 15 min in the dark, cells were exposed to
ionizing radiation. 8-Hydroxy-20 -deoxyguanosine (8-OH-dG) levels of U937
cells were estimated by using a fluorescent binding assay as described by
Struthers et al. (18). After U937 cells were exposed to ionizing radiation,
cells were fixed and permeabilized with ice-cold methanol for 15 min. DNA
damage was visualized with avidin-conjugated TRITC (1:200 dilution) for
fluorescent microscope with 540 nm excitation and 588 nm emission.
Mice
Adult male ICR mice were obtained from Hochang Science (Taegu, Korea).
The animals were housed five per cage in climate-controlled, circadian
rhythm-adjusted 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 ref. 9.
Mitochondrial damage
Mitochondrial membrane potential transition (MPT) was measured by the
incorporation of rhodamine 123 dye into the mitochondria, as described previously (19). Cells (1 106 ) grown on poly-L-lysine coated slide glasses were
exposed to ionizing radiation. Cells were then treated with 5 mM rhodamine
123 for 15 min and excited at 488 nm with an argon laser. Cells were doublestained with 100 nM MitoTracker Red, which is a morphological marker of
mitochondria. The fluorescence images at 520 nm were simultaneously
obtained with a laser confocal scanning microscope. To evaluate the levels of
mitochondrial ROS U937 cells in PBS were incubated for 20 min at 37 C with
5 mM DHR 123 and cells were double-stained with 100 nM MitoTracker Red.
Cells were washed, resuspended in complete growth media, and ionizing
radiation was applied to the cells. The cells were then incubated for an additional 40 min. DHR 123 and Mitracker Red fluorescence were visualized by a
fluorescence microscope. Intracellular ATP levels were determined by using
luciferin-luciferase as described (20). Light emission was quantified in a Turner
Designs TD 20/20 luminometer (Stratec Biomedical Systems, Germany).
Toxicology of NtBHA and whole-body irradiation
To determine its maximal tolerated dose, solutions of NtBHA were freshly
prepared in saline. Two groups of 15 mice each received either NtBHA or
saline. NtBHA was administered prior to irradiation at a dose of 5 mg/kg in
volumes equivalent to 1% of each animal's weight once daily for 2 weeks.
Control mice were given saline and all injections were administered intraperitoneally. Survival was assessed up to 30 days after injection without irradiation. To determine survival after whole-body irradiation, the same protocol
for the NtBHA 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 137 Cs
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 of mice were homogenized with a solution of 0.3 M
sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 mM leupeptin
and 100 mg/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 ÿ20 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.
Cellular redox status
NADPH was measured using the enzymatic cycling method as described by
Zerez et al. (10) 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 (e ˆ 1.36 104 M ÿ1 cm ÿ1 ) as
described by Akerboom and Sies (11), and GSSG was measured by the
DTNB-GSSG reductase recycling assay after treating GSH with 2-vinylpyridine (12). 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 mM CMAC cell tracker for 30 min. For in vivo visualization
of GSH levels in tissues, tissues were perfused with 5 mM CMAC for 60 min
and the cryosections of tissues were prepared. The images of CMAC cell
tracker fluorescence by GSH were analyzed by the Zeiss Axiovert 200 inverted
microscope at fluorescence DAPI region (excitation, 351 nm; emission,
380 nm) (13). Intracellular ROS production was measured using the oxidantsensitive fluorescent probe DCFH-DA with fluorescence-activated cell
sorter (FACS) analyses. Measurement of 20 ,70 -dichlorofluorescein (DCF)
fluorescence in cells was made at least 10 000 events/test using a FACS
caliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with a fluorescein isothiocyanate filter. For in vivo visualization of ROS generation in
tissues, tissues were perfused with 5 mM DCFH-DA for 25 min. The DCF
fluorescence from cryosections of tissues was observed with a fluorescence
microscope. Intracellular hydrogen peroxide concentrations were determined
using a ferric-sensitive dye, xylenol orange, as described (14).
Cellular oxidative damage
The protein carbonyl content was determined spectrophotometrically using the
DNPH labeling procedure as described (15). The protein carbonyl contents in
U937 cells also determined with DNP-specific antibody (1:200 dilution) and
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Quantification of relative fluorescence
The averages of fluorescence intensity from fluorescence images were calculated as described (21).
Statistical analysis
The difference between two mean values was analyzed by Student's t-test and
was considered to be statistically significant when P 5 0.05.
Results
Cell killing
When cultured U937 cells were exposed to g-irradiation, a
dose-dependent decrease in cell viability was observed. However, the cells pre-treated with 0.1 mM NtBHA for 2 h were
significantly more resistant than control cells untreated with
NtBHA (Figure 1). The protective effect of NtBHA was concentration-dependent and NtBHA itself up to 1 mM was without effect on the viability of U937 cells (data not shown). The
protective effect of NtBHA against ionizing radiation was also
confirmed by dual staining with calcein-AM and ethidium
homodimer-1. Ionizing radiation caused lethal injury to
U937 cells and their nuclei were mostly stained with ethidium
homodimer-1 to exhibit a red fluorescence. NtBHA (0.1 mM)
pre-treatment for 2 h decreased the proportion of red fluorescent nuclei of dying cells in g-irradiated cultures (data not
shown).
Cellular redox status
To investigate whether the difference in viability of U937 cells
upon exposure to ionizing radiation is associated with ROS
formation, the levels of intracellular peroxides in the
U937 cells were evaluated by FACS analyses with the
oxidant-sensitive probe DCFH-DA. As shown in Figure 2(A),
an increase in DCF fluorescence was observed in U937 cells
when they were exposed to 20 Gy of g-irradiation. The
N-t-butyl hydroxylamine as a radioprotector
Fig. 1. Resistance of control (filled circle) and NtBHA-treated (filled
triangle) U937 cells against ionizing radiation. Cells (2 105 /well) were
grown in 96-well plates and then exposed to different doses of g-irradiation
prior to measurement of cell viability by MTS assay. Cell viability is
expressed as a percentage of absorbance seen in the untreated control cells.
Each value represents the mean SD from five independent experiments.
increase in fluorescence was significantly reduced in cells pretreated with 0.1 mM NtBHA for 2 h. The pre-treatment of
NtBHA also resulted in a significantly lower intracellular level
of H2 O2 as compared with that of untreated control with the
exposure of 20 Gy of irradiation (Figure 2B). These data
strengthen the conclusion that NtBHA provided protection
from the cytotoxic actions of g-irradiation by decreasing the
steady-state level of intracellular oxidants. GSH is one of the
most abundant intracellular antioxidants and determination of
changes in its concentration provides an alternative method of
monitoring oxidative stress within cells. Cellular GSH levels
determined with the GSH-sensitive fluorescent dye CMAC in
U937 cells treated with 20 Gy of g-irradiation were significantly decreased (Figure 3A). However, the depletion of GSH
was significantly protected by the pre-treatment of 0.1 mM
NtBHA 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 g-irradiation, the ratio of cellular [GSSG]/[GSHt ]
was significantly higher in control cells than in NtBHA-treated
cells (Figure 3B). These data indicate that GSSG in control
cells was not reduced as efficiently as in NtBHA-treated cells.
Fig. 2. Effects of NtBHA on peroxide level and ROS generation. (A) Effect of NtBHA on g-irradiation-induced ROS production. Cells were exposed to
g-irradiation and subjected to FACS analyses. (B) Production of hydrogen peroxide in U937 cells was determined by the method described under Materials and
methods. Each value represents the mean SD from five independent experiments. P 5 0.01 compared with irradiated control cells.
Fig. 3. Effect of NtBHA on the cellular redox status of U937 cells exposed to ionizing radiation. (A) Effect of NtBHA on GSH levels. Fluorescence image
of CMAC-loaded cells was obtained under microscopy. The averages of fluorescence intensity were calculated as described (21). Each value represents the mean
SD from three independent experiments. (B) Ratios of GSSG versus total GSH pool. Each value represents the mean SD from three independent experiments.
#
(C) Ratios of NADPH versus total NADP pool. Each value represents the mean SD from three independent experiments. P 5 0.05 and P 5 0.01 compared
with irradiated control cells.
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J.H.Lee, I.S.Kim and J.-W.Park
Fig. 4. Cell damage of U937 cells. (A) Lipid peroxidation of U937 cells after exposure to ionizing radiation. The level of MDA accumulated in the cells
unexposed and exposed to g-irradiation was determined by using a TBARS assay. Each value represents the mean SD from four independent experiments.
(B) Protein carbonyl content of U937 cells exposed to ionizing radiation. Protein carbonyls were measured in cell-free extracts by the method of Levine et al. (15)
with the use of DNPH. Each value represents the mean SD from five independent experiments. (C) Relative intensities of DNP-FITC, HNE-TRITC, DPPP
and avidin-TRITC fluorescences in U937 cells. Protein carbonyl content of U937 cells detected with DNP-specific antibody and anti-human IgG FITC
conjugate as a secondary antibody. Lipid peroxidation in U937 cells was detected with polyclonal anti-HNE Michael adduct antibody and anti-human IgG TRITC
conjugate. To visualize lipid peroxidation in U937 cells exposed to g-irradiation, cells (1 106 /ml) were stained with 5 mM DPPP for 15 min. Fluorescence
images were obtained under microscopy. In order to estimate 8-OH-dG levels in U937 cells, the cells were fixed and permeabilized immediately after exposure to
g-irradiation. 8-OH-dG levels reflected by the binding of avidin-TRITC were visualized by a fluorescence microscope. The averages of fluorescence intensity
were calculated as described (21). Each value represents the mean SD from three independent experiments. P 5 0.01 compared with irradiated control cells.
NADPH, required for GSH generation by glutathione reductase, is an essential factor for the cellular defence against
oxidative damage. The ratio for [NADPH]/[NADP ‡ ‡
NADPH] was significantly decreased in cells treated with 20
Gy of g-irradiation, however, the decrease in this ratio was
much less pronounced in NtBHA-treated cells (Figure 3C).
Cellular oxidative damage
As indicative markers of oxidative damage to cells, the occurrence of oxidative DNA damage, protein oxidation and lipid
peroxidation were evaluated. 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 (22). Exposure of
20 Gy of g-irradiation increased the level of MDA 5-fold in
control cells, however, the increase in MDA content of
NtBHA-treated cells was significantly lower than that of
untreated cells (Figure 4A). To determine whether NtBHA
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pre-treatment decreased the sensitivity to protein damage, we
performed carbonyl content measurements for protein oxidation after cellular exposure to ionizing radiation. Control cells
elicited an ~2-fold increase of carbonyl groups, as compared
with unirradiated cells when 20 Gy of ionizing radiation was
exposed. Although the carbonyl content of NtBHA-treated
cells also increased with irradiation, the increase was significantly lower than that of control cells (Figure 4B). Protein
oxidation was also visualized by immunocytochemical method
using anti-DNP antibody. As shown in Figure 4(C), the fluorescent intensity, which reflects the endogenous levels of
carbonyl groups in proteins was significantly increased when
U937 cells were exposed to 20 Gy of g-irradiation and the
increase of protein oxidation was markedly reduced in
NtBHA-treated cells. Lipid peroxidation was visualized by
immunocytochemical method using anti-HNE antibody. The
fluorescent intensity, which reflects the endogenous levels of
HNE adducts in proteins was significantly increased when
U937 cells were exposed to 20 Gy of g-irradiation and the
N-t-butyl hydroxylamine as a radioprotector
Fig. 5. Effects of NtBHA on mitochondrial function. (A) Effect of NtBHA on MPT. MPT of U937 cells was measured by the incorporation of rhodamine
123 dye into the mitochondria. (B) Effect of NtBHA on mitochondrial ROS generation. DHR 123 was employed to detect mitochondrial ROS. DHR 123
fluorescenec was visualized by a fluorescence microscope. The averages of fluorescence intensity from fluorescence images were calculated as described (21).
Each value represents the mean SD from three independent experiments. (C) Effect of NtBHA on the levels of intracellular ATP. Control and NtBHA-treated
U937 cells were irradiated and assayed for intracellular ATP content. Each value represents the mean SD from five independent experiments. P 5 0.01
compared with irradiated control cells.
increase of lipid peroxidation was markedly reduced in
NtBHA-treated cells. Recently, it has been shown that DPPP
is a suitable fluorescence probe to monitor lipid peroxidation
within cell membrane specifically (17). DPPP fluorescent
intensity was increased markedly in untreated cells, whereas
it was increased slightly in NtBHA-treated cells after exposure
to ionizing radiation. 8-OH-dG, the most abundant and most
studied lesion in DNA generated by intracellular ROS, has
been used as an indicator of oxidative DNA damage in vivo
and in vitro (23). Recently, it has been shown that 8-OH-dG
level is specifically measured by a fluorescent binding assay
using avidin-conjugated TRITC (18). The fluorescent intensity, which reflects the endogenous levels of 8-OH-dG in DNA
was significantly increased in untreated cells upon exposure to
ionizing radiation. In contrast, the overall DNA appeared to be
markedly protected in NtBHA-treated cells even after exposure to the same dose of ionizing radiation. These results
indicate that NtBHA appears to protect cells from oxidative
damage caused by ionizing radiation.
Mitochondrial damage
MPT is a very important event in both necrosis and apoptosis,
and ROS is one of the major stimuli that change MPT (24). To
answer whether NtBHA modulates the MPT upon exposure to
ionizing radiation, 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 NtBHA-treated cells (Figure 5A). The intracellular
peroxide levels in the mitochondria of U937 cells were evaluated by confocal microscopy with the oxidant-sensitive
probe DHR 123. As shown in Figure 5(B), the intensity of
fluorescence was significantly lower in cells pre-treated with
0.1 mM NtBHA for 2 h when compared with that in the
mitochondria of untreated cells upon exposure to 20 Gy of
g-irradiation. Mitochondrial injury is often followed by the
depletion of intracellular ATP level. As shown in Figure 5(C),
the decrease in ATP level was markedly prevented in NtBHAtreated cells when U937 cells were exposed to 20 Gy of
g-irradiation.
Fig. 6. Toxicity of NtBHA and effects of NtBHA on the radiation sensitivity
of ICR mice. The survival of mice administered with NtBHA at 5 mg/kg
daily for 2 weeks without irradiation (filled square) was observed over
30 days. Groups of 15 ICR mice received an injection of either NtBHA
(5 mg/kg) i.p. (filled triangle) or saline (filled circle) for 2 weeks, were
exposed to 8 Gy of whole-body irradiation, and were then observed over
30 days for survival.
In vivo radioprotection
ICR male mice treated with NtBHA at 5 mg/kg daily for 2
weeks survived with no apparent adverse effects after 30 days.
Mice were injected intraperitoneally with saline or NtBHA at
5 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 displayed in Figure 6. NtBHA administration before irradiation provided significant protection compared with control animals receiving saline. The kidneys
from NtBHA-administered mice prior to g-irradiation showed
the lower level of hydrogen peroxide (Figure 7A) compared
with mice not administered NtBHA. The ratio for [GSSG]/
[GSHt ] was lower (Figure 7B) and the ratio for [NADPH]/
[NADPH ‡ NADP ‡ ] was higher (Figure 7C) in the kidneys of
NtBHA-administered mice before g-irradiation. The livers and
lungs showed the similar results (data not shown). In the
kidneys of untreated mice, TBARS and carbonyl groups in
proteins were increased significantly after whole-body irradiation. Administration of NtBHA markedly prevented the
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J.H.Lee, I.S.Kim and J.-W.Park
Fig. 7. Effects of NtBHA on redox status of the kidneys from mice (n ˆ 3--5/group) exposed to ionizing radiation. (A) Production of hydrogen peroxide in the
kidneys from mice was determined as described under Materials and methods. Each value represents the mean SD from five independent experiments.
(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. P 5 0.05 and P 5 0.01 compared with irradiated control mice.
Fig. 8. Effects of NtBHA on oxidative damage of the kidneys from mice (n ˆ 3--5/ group) exposed to ionizing radiation. (A) The levels of MDA accumulated in
the kidneys from mice exposed to g-irradiation. Each value represents the mean SD from three independent experiments. (B) Protein carbonyl content of
kidneys after ionizing radiation in mice received an injection of either NtBHA or saline. Each value represents the mean SD from three independent
experiments. (C) Changes in the ATP content of the kidneys after g-irradiation. Control and NtBHA-treated mice were irradiated and assayed for intracellular
#
ATP content from kidneys. Each value represents the mean SD from five independent experiments. P 5 0.05 and P 5 0.01 compared with irradiated
control mice.
increase in TBARS and carbonyl groups in proteins (Figure 8A
and B). As shown in Figure 8(C), the kidneys from non-NtBHAadministered mice showed the lower level of ATP compared
with untreated mice, suggesting the role of NtBHA against the
loss of mitochondrial function caused by ionizing radiation. The
livers and lungs showed the similar results (data not shown).
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
cells by ionizing radiation is due to formation of hydroxyl
radicals, which reacts with almost all cellular components to
induce oxidative damage to DNA, lipid and protein (3,25,26).
DNA is a particularly important target, suffering double- and
single-strand breaks, deoxyribose damage and base modification (27). Of the total damage to DNA caused by ionizing
radiation, as much as 80% may result from radiation-induced
water-derivative free radicals and secondary carbon-based
1440
radicals (1). In addition to the generation of hydroxyl radicals,
the hydrated electrons formed by ionizing radiation can reduce
it to superoxide. Superoxide can dismutate to hydrogen peroxide with the possibility of additional hydroxyl radicals 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 ionizing radiation. 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 hydrogen radical (H) from thiols
must not be ignored (27). Recently, it has been reported that
several non-thiol compounds including tempol (4), flavonoids
(28) and Zn-desferrioxamine (29) are identified as potential
radioprotectors.
The spin-trap PBN has been shown to protect against oxidative damage in different biological models (30--32). NtBHA
and benzaldehyde are the breakdown products of PBN.
NtBHA reacts with ROS and reactive aldehydes, and thus
could prevent oxidative damage and lipid peroxidationmediated damage to cellular macromolecules (33). NtBHA
N-t-butyl hydroxylamine as a radioprotector
delays senescence of IMR90 cells at 20 times lower
concentration compared with PBN to produce a similar
effect. Recently, we have shown that PBN is an effective
radioprotector in vivo and in vitro (6). Therefore, it was plausible to assume that NtBHA may play a role in preventing
oxidative damage caused by ionizing radiation in cells and
animals at the much lower concentrations compared with
PBN. The aim of the present work was to evaluate the role of
NtBHA in protecting U937 cells and mice from ionizing radiation in regards 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 defence mechanisms including antioxidant enzymes and small molecular weight antioxidants to
cope with lethal oxidative environments. GSH is known to
play a role in protecting cells against ionizing radiation. Treatment with buthionine sulfoximine to inhibit GSH synthesis
increases radiosensitivity (34). The depletion of intracellular
GSH and the increase in the ratio of [GSSG]/[GSHt ], which
reflects the efficiency of GSH recycling were significantly
reduced by NtBHA. The ratio for [NADPH]/[NADP ‡ ‡
NADPH], the other parameter that reflects the cellular redox
status and the availability of the reducing equivalent for GSH
turnover by glutathione reductase, was significantly increased
by NtBHA. These results indicate that ionizing radiation
results in the perturbation of cellular redox balance presumably by depletion of GSH and NADPH pools and NtBHA may
shift the balance to antioxidant condition. Whether the presence of NtBHA induced concomitant alterations in the activity of major antioxidant enzymes was investigated. The
activity of antioxidant enzymes such as SOD, catalase,
glutathione peroxidase, glucose 6-phosphate dehydrogenase
and glutathione reductase was decreased upon exposure to girradiation, however, NtBHA did not significantly affect the
activity of antioxidant enzymes (data not shown). These results
indicate that NtBHA may act as a direct scavenger of ROS.
Mitochondria are vulnerable to oxidants because they are the
major source of free radicals in the cells and are limited in their
ability to cope with oxidative stress (35). Therefore, improving
the mitochondrial status could cause a significant decrease in
the level of oxidants in the cells (8). It is well established that
mitochondrial dysfunction is directly and indirectly involved
in a variety of pathological states. All the changes caused by
ionizing radiation are compatible with mitochondrial failure,
encompassing reduced production of ATP, generation of ROS,
and accumulation of rhodamine 123, which reflect mitochondrial swelling or changes in the mitochondrial inner membrane. It has been suggested that mitochondria are a potential
primary target for NtBHA in delaying senescence of IMR90
cells (8). NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radicalinduced toxicity to mitochondria (33). A clear suppression of
such damages indicates that NtBHA prevents a deterioration of
the bioenergetic state.
NtBHA is not only an in vitro but in vivo radioprotector.
Radioprotective effect of NtBHA reflected by suppression of
lethality was evident 30 days after exposure to radiation. The
measurement of lipid peroxidation and protein oxidation in
kidneys, livers and lungs from mice exposed to g-irradiation
indicates that the damage caused by ionizing radiation was
similar in these tissues and that NtBHA protects damage in
tissues in a similar manner. To confirm oxidative damage in
irradiated mice due to the pro-oxidant status, the formation of
ROS in tissues was measured. NtBHA administration showed
the tendency to lower the formation of ROS. The observed
beneficial effects of NtBHA in mice supported here offer the
possibility of developing antioxidant approaches to treating
damage caused by ionizing radiation. The use of NtBHA can
avoid benzaldehyde, which is both mutagenic and carcinogenic (36), formed when PBN decomposes. In conclusion,
NtBHA is effective in protecting cells and mice from oxidative
stress caused by ionizing radiation and alleviated damage
suggests that further study of NtBHA or similar compounds
is warranted.
Acknowledgement
This work was supported by the Korea Research Foundation Grant
(KRF-2003-041-E00221).
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Received December 24, 2003; revised February 9, 2004;
accepted February 27, 2004