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Apoptosis 2004; 9: 757–763
C
2004 Kluwer Academic Publishers
Effects of antioxidants on X-ray- or
hyperthermia-induced apoptosis in human
lymphoma U937 cells
Z.-G. Cui, T. Kondo, L. B. Feril, Jr., K. Waki, O. Inanami and M. Kuwabara
Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University,
2630 Sugitani, Toyama 930-0194 (Z.-G. Cui, T. Kondo, L. B. Feril, Jr.); Laboratory of Radiation Biology, Department of
Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818,
Japan (K. Waki, O. Inanami, M. Kuwabara)
Hydroxyl radicals (·OH) and superoxide anion radicals
(O ·−
2) are known to play cardinal roles in cell killing and
various types of cell damage. In order to elucidate the
mechanism of the involvement of both free radicals on
apoptosis, the correlation between anti-apoptotic effects
and free radical scavenging abilities of anti-oxidants was
studied. As an indicator of anti-apoptotic effects, C1/2
(antioxidant concentration to inhibit DNA fragmentation
by 50%) was evaluated in human lymphoma cell line U937
cells 6 hr after X-ray (10 Gy) or hyperthermia (44◦C, 30
min) treatment. Rate constants of the reactions between
antioxidants and ·OH or O ·−
2were calculated as the scav-
enging ability of the antioxidants with graded concentra-
tion estimated by EPR spectroscopy. No apparent cor-
relation between C1/2 obtained in apoptosis induced by
X-rays or hyperthermia and the rate constants of antiox-
idants for ·OH or O ·−
2was observed. On the other hand,
the partition coefficients in 1-octanol/water of the antiox-
idants, an indicator of hydrophobicity, revealed a corre-
lation with the C1/2 of the agents with hyperthermia, but
not with X-ray irradiation. These results indicate that the
prevention of apoptosis by an antioxidant is not simply
associated with its scavenging ability for ·OH or O ·−
2. The
hydrophobicity of the antioxidant, among other possible
factors, is involved in the inhibition of hyperthermia- in-
duced apoptosis.
Keywords: antioxidant; apoptosis; hyperthermia; X-rays.
Introduction
It is known that primary reactive oxygen species (ROS),
such as hydroxyl radicals (·OH) and superoxide anion rad-
icals (O ·−
2)are constantly produced in cells as a conse-
quence of aerobic metabolism and play a major role in
Correspondence to: Prof. Takashi Kondo, Department of Ra-
diological Sciences, Faculty of Medicine, Toyama Medical
and Pharmaceutical University, 2630 Sugitani, Toyama 930-
0194, Japan. Tel.: +81-76-434-7265; Fax: +81-76-434-5190;
e-mail: kondot@ms.toyama-mpu.ac.jp
a variety of mammalian biological processes including
homeostatic function, immunological defense and the sig-
nal transduction system of cells. These free radicals subse-
quently generate hydrogen peroxide (H2O2)and lipid per-
oxides which are relatively stable. These oxidative stresses
caused by ROS play a critical role in the induction of
apoptosis.1,2
Ionizing radiation is a well-known physical factor that
can generate ·OH radicals due to radiolysis of water
molecules. This free radical rapidly reacts with many bi-
ological macromolecules, such as nucleic acids, proteins
and lipids and induce nucleic base damage, single- and
double-strand breaks of DNA, DNA-protein cross-links,
lipid peroxidation and protein degradation.3,4The bio-
logical effects of ionizing radiation are mostly due to DNA
damage, and membrane damage such as lipid peroxida-
tion. Both DNA and membrane damage play important
roles in radiation-induced apoptosis.5,6In addition, it has
been shown that hyperthermia induces intracellular O ·−
2
generation.7,8We also reported that the enhancement of
hyperthermia-induced apoptosis by some agents are as-
sociated with increase in intracellular O ·−
2formation in
human lymphoma U937 cells.9,10
The sensitivity of cells to oxidative stress depends on its
inherent antioxidation and detoxification mechanisms.11
Exogenous antioxidants can also protect cells against ox-
idative stress involved in apoptosis. For example, the
antioxidant N-acetyl-cysteine (NAC) can serve as a pre-
cursor of the endogenous antioxidant glutathione to pro-
tect hydroquinone-induced apoptosis in human embry-
onic kidney cells and also against ultrasound-induced
apoptosis in U937 cells.12–14 Another antioxidant cime-
tidine suppresses X-ray-induced micronuclei and apopto-
sis via ·OH scavenging15 and a water-soluble derivative
of vitamin E, trolox, prevents X-ray- or hyperthermia-
induced apoptosis by inhibiting lipid peroxidation.16,17
Two known mechanisms are involved in the anti-apoptotic
action of antioxidants. One is the scavenging of ROS and
Apoptosis ·Vol 9 ·No6·2004 757
Z.-G. Cui et al.
the other is by stimulating the cellular defense system
against oxidative stress.18,19 But the details underlying
these mechanisms are still not entirely clear.
In the present study, to further investigate the roles of
·OH and O ·−
2in apoptosis, the relationship between the
ability of antioxidants to scavenge ·OH and O ·−
2, and
the inhibition of X-ray- or hyperthermia-induced apop-
tosis was studied. In addition, as an indicator of the hy-
drophobicity of the antioxidant, the partition coefficient
was determined to examine the role of the membrane-
antioxidant interaction on the anti-apoptotic efficiency of
these agents.
Materials and methods
Cells and cell culture
The human myelomonocytic lymphoma cell line, U937,
was obtained from the Human Sciences Research Resource
Bank, (Human Sciences Foundation, Tokyo, Japan). The
cells were grown in RPMI 1640 culture medium (Invit-
rogen, Groningen, The Netherland) supplemented with
10% heat-inactivated fetal bovine serum (Gibco, Carls-
bad, CA) at 37◦Cinhumidified air with 5% CO2. Cells
in log-phase (doubling time is 23.5 hr) were used for ex-
periments, after confirmation that they were free of any
mycoplasma contamination.
Antioxidant chemicals
In the present study, we used ten kinds of the antioxi-
dants. All of these antioxidants have been reported to have
anti-apoptotic activity. α-phenyl-N-tert-butyl-nitrone
(PBN), α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone
(POBN), 2,2,6,6-tetrametyl-1-piperidinyl-1-oxyl (Tem-
pol), 4-hydroxy-2,2,6,6,-tetramethylpiperidine-N-oxyl
(Tempo), and 6-hydroxyl-2,5,7,8-tetramethyl-chroman-
2-carboxylic acid (Trolox) were purchased from Aldrich,
Milwaukee, WI.; 3-methyl-1-phenyl-pyrazoline-5-one
(Edaravone) was kindly provided by Mitsubishi-Pharma
Corp. Osaka, Japan; N-acetyl-L-cysteine (NAC),
cimetidine, cysteamine hydrochloride, 3(2)-tert-butyl-
4-hydroxyanisole (BHA) and the other reagents were
obtained from Wako Pure Chemical Industries Ltd.,
Osaka, Japan; the agent, 5,5-dimethyl-1-pyrroline
1-oxide (DMPO) was purchased from LABOTEC Co.
Ltd., Tokyo, Japan. To prepare the stock solutions of the
antioxidants, Trolox was dissolved in 1.0 M of NaHCO3
at a concentration of 300 mM and the pH was adjusted
to 7.0 using HCl. BHA was dissolved in DMSO (the
final concentrations of DMSO during use was always less
than 1%). Edaravone was dissolved in 0.1 N NaOH at a
concentration of 500 mM. The other antioxidants were
directly dissolved in the culture medium just before
use.
Exposure to X-ray or hyperthermia
Freshly prepared antioxidant at concentrations 0.0, 1.0,
2.0, 5.0 and 10.0 mM was added to the cell pellet contain-
ing about 3×106cells/ml and then irradiated at a dose of
10 Gy or exposed to hyperthermia at 44.0◦C for 30 min af-
ter about 15 minutes incubation. A 6-cm diameter plastic
culture dish that contains 4 ml of the sample was prepared
for X-irradiation. X-irradiation was carried out at room
temperature by an X-ray apparatus (MBR-1520R-3, Hi-
tachi Medico Technology Co., Kashiwa, Japan) operating
at 150 kV and 20 mA at a dose rate of 5 Gy/min de-
termined by Fricke dosimetry. Hyperthermic treatments
were performed by immersing plastic culture tubes con-
taining culture medium (3 ml) in a water-bath (NTT-
1200, Eyela, Tokyo, Japan) at 44.0±0.05◦C. The tem-
perature of the solution inside the flask or test tube was
monitored with a digital thermometer (#7563, YOKO-
GAWA, Tokyo, Japan) coupled with a thermocouple 0.8
mm in diameter during heating. After the treatment the
cells were then incubated with the antioxidant in 5% CO2
at 37◦Cfor6hr.
DNA fragmentation assay
The amount of DNA extracted from cells that had under-
gone DNA fragmentation was assayed using the method
of Sellins and Cohen20 with a few modifications.21 Briefly,
about 3×106cells were lysed using 200 µloflysis buffer
(10 mM Tris, 1 mM EDTA, 0.2% Triton X-100, pH 7.5)
and centrifuged at 13,000 g for 10 min. Subsequently,
each DNA sample in the supernatant and the result-
ing pellet were precipitated in 12.5% trichloroacetic acid
(TCA) at 4◦C, and quantified using the diphenylamine
reagent after hydrolysis in 5% TCA at 90◦C for 20 min.
The percentage of fragmented DNA for each sample was
calculated as the amount of DNA in the supernatant di-
vided by the total DNA for that sample (supernatant plus
pellet).
Assessment of apoptosis by flow cytometry
Flow cytometry was performed with propidium iodide
(PI) and fluorescein isothiocyanate (FITC)-labeled an-
nexin V to detect Phosphotidylserine (PS) externaliza-
tion of apoptosis. The cells were washed twice in cooled
phosphate-buffered saline, and adjusted to 106cells/ml
with the biding buffer of the Annexin V-FITC kit (Im-
munotech, Marseille, France). FITC-labeled annexin V
(5 µl) and propidium iodide (PI) (5 µl) were added to
758 Apoptosis ·Vol 9 ·No6·2004
Antioxidant and apoptosis
the suspension (490 µl) and mixed gently. After incuba-
tion for 10 min in the dark, the cells were analyzed with a
flow cytometer (EPICS XLTM, Beckman-Coulter, Miami,
FL)22.
EPR measurement of rate constants
forhydroxyl radicals
Electron paramagnetic resonance (EPR)-spin trapping
with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was
utilized for the measurement of rate constants of antiox-
idants for reactions of ·OH induced by X-rays. Aqueous
DMPO solutions at a concentration of 10 mM saturated
with air containing various concentrations of ·OH scav-
engers were exposed to X-rays (150 Gy). EPR spectra
of irradiated samples were measured in a quartz flat cell
with an EPR spectrometer (RFR-30 Radical Analyzer Sys-
tem, Radical Research Co., Tokyo, Japan). The relation-
ship curve between the rate constants and the C1/2 values
for the water soluble ·OH scavengers potassium iodide,
sodium formate, mannitol, glucose, sodium propionate
and sodium acetatewas utilized as standard.15,23 C1/2 is
the concentration of the scavenger at which the DMPO-
OH adduct is decreased by 50% of the maximum yield.
The rate constants of the antioxidants used in this study
were then calculated based on the measured C1/2 values
and the above standard.
EPR measurement of rate constants
for superoxide
The rate constant for the reaction of each drug with O ·−
2
was determined in competition experiments using the
spin trap, DMPO. Each drug was prepared in a reaction
mixture that contained 0.5 mM hypoxanthine, 0.1 M 3-
morpholinopropanesulfonic acid (pH 7.4), 0.1 mM di-
ethylenetriaminepentaacetic acid, 20 µU/ml xanthine ox-
idase. After 200 µlofreaction mixture was prepared,
the sample solution was immediately transferred to a flat
quartz EPR cell (LLC-04B, Labotec Co., Tokyo, Japan)
and EPR spectra were recorded at room temperature by
an X-band EPR spectrometer (RE-1X, JEOL Co., Tokyo,
Japan). The EPR signal intensity (MI=+1) of DMPO-
OOH was measured at 5 min after start of the reaction.
The rate constant of each drug was calculated as fol-
lows; when the rate constants of DMPO and drug A for
O·−
2are designated as k1and k2, respectively, the com-
petition kinetics of drug A against DMPO are given by
the following equation:(DMPO-OOH)0/(DMPO-OOH)
=1+k2[drugA]/k1[DMPO] where (DMPO-OOH)0is
the signal intensity of DMPO-OOH in the absence of drug
A, (DMPO-OOH) is the signal intensity of DMPO-OOH
in the presence of drug A, and [DMPO] and [drug A] are
the concentrations of DMPO and drug A, respectively. By
plotting {(DMPO-OOH)0/ (DMPO-OOH)}-1 against
[drug A]/[DMPO], a linear relationship is obtained in
which the slope of the straight line is equal to the ratio
of the rate constants, k1/k2. Since the rate constant (k1)
of DMPO with O ·−
2is reported as 30 M−1s−1at pH 7.4
from a previous study,24 the rate constant (k2)of drug A
with O ·−
2can be calculated.
Measurement of partition coefficients
As a measure of the hydrophobicity of the antioxidants,
the partition coefficients of the antioxidants were de-
termined, K =Co/Cw, where Co is the concentration
of the antioxidants in 1-octanol and Cw is the con-
centration of the antioxidants in water. The concentra-
tions of the antioxidants in 1-octanol and water were
measured spectrophotometrically after attaining equilib-
rium by continued shaking. The phase was separated by
centrifugation.
Results
Hyperthermia- and X-ray-induced apoptosis was mea-
sured by DNA fragmentation and flow cytometry. U937
cells were treated by hyperthermia at 44◦C for 0, 10, 20
and 30 min or X-ray at doses of 0, 5, 10 and 20 Gy and
incubated in 5% CO2at 37◦C for 6 hr after the treatment.
The results showed that with hyperthermia the DNA frag-
mentation rates were: 7.3 ±1.3%, 21.8 ±3.9%, 35.4
±3.3% and 60.6 ±2.9% (n=6) for the 0, 10, 20,
and 30 min treatments, respectively. With flow cytom-
etry, early apoptosis were: 1.0% ±0.4%, 8.6 ±3.3%,
41.4 ±5.8% and 60.9 ±5.1% (n=7), in the same
order. The cells treated by X-ray revealed 6.7 ±1.3%,
9.2 ±1.0%, 29.8 ±4.9% and 54.0 ±6.5% (n=7)
of DNA fragmentation, and 0.6 ±0.1%, 1.8 ±0.7%,
22.6 ±3.6% and 59.8 ±2.1% (n=3) of early apopto-
sis for the doses 0, 5, 10 and 20 Gy, respectively. In any
of the treatments, secondary necrosis was not changed
significantly. These results indicate that measurement of
apoptosis by DNA fragmentation is sufficiently reflective
of the actual amount of apoptosis induced by either hy-
perthermia at 44◦C for 30 min or X-ray at 10 Gy. In
succeeding experiments we utilized DNA fragmentation
assays for apoptosis measurements.
It is known that ·OH is one of the most reactive radi-
cal involve in X-ray-induced cell killing,5,6while O ·−
2is
involved with hyperthermia-induced apoptosis.9,10 In the
present study, the antioxidants scavenged ·OH generated
by X-irradiation in a dose dependent manner (Figure 1).
The rate constant of ·OH for the antioxidants was esti-
mated by comparing the relationships between the C1/2
values for the agents, potassium iodide, sodium formate,
Apoptosis ·Vol 9 ·No6·2004 759
Z.-G. Cui et al.
Figure 1. Scavenging of ·OH by the antioxidants. The solutions
of the antioxidants were prepared at the indicated concentrations
before the X-irradiation at a dose of 150 Gy. The scavenging of
·OH was measured immediately after the irradiation by EPR using
the spin trapping agent DMPO as described in the material and
methods. Points in the graph are the averages of the results of
four experiments.
mannitol, glucose, sodium propionate and sodium acetate
which are water-soluble ·OH scavengers and these rate
constants.15 The anti-apoptotic effect of the antioxidant
on the apoptosis induced by X-ray (10 Gy) or hyperther-
mia (44.0◦C30min), were examined by DNA fragmen-
tation, and the C1/2 values of the each of the antioxidants
was evaluated. When we compared the rate constants and
the C1/2 values, the results showed no significant corre-
lation between the C1/2 values and the rate constants for
reactions of ·OH both in apoptosis induced by X-ray and
hyperthermia. Even edaravone and NAC did not have
anti-apoptotic effect despite of their high rate constant
for ·OH. These results indicate that the anti-apoptotic
effect is not solely due to the scavenging ability of the an-
tioxidants of ·OH both in apoptosis induced by X-ray or
hyperthermia (Figure 2A and B). In addition, the relation-
ship between the rate constants for reaction of O ·−
2of the
antioxidants were also evaluated and the relationship with
C1/2 were investigated. The results showed there was also
no notable correlation between the rate constant for O ·−
2
and the C1/2 values both in apoptosis induced by X-ray
or hyperthermia (Figure 3A and B). These results point to
the conclusion that scavenging ability of the antioxidants
on O ·−
2was also not the only factor for the prevention of
the apoptosis induced by X-ray and hyperthermia.
Since ROS scavenging activity alone could not explain
the anti-apoptotic effects of the antioxidants, other possi-
ble factors were considered. Considering the importance
of the cell membrane in regulating interactions and up-
take of exogenous compounds, the hydrophobicity of the
antioxidants were determined. As an indicator of the hy-
Figure 2. Relationship between rate constants of antioxidants
for ·OH and C1/2 for apoptosis induced by X-ray or hyperther-
mia. U937 cells were irradiated at doses of 10 Gy (A) or treated
with 44.0◦Cfor 30 min (B) in the presence or absence of the
antioxidants; the cells were harvested 6 hr after the treatment
and the rate of the DNA fragmentation was measured. The rate
constants of reaction for ·OH were evaluated as described in the
material and methods. Values above 14 mM along Y-axis were not
shown in the figure. The data are the averages of four independent
experiments.
drophobicity of the antioxidant, the partition coefficient
was measured. The results showed that the hydrophobic-
ity of the antioxidants was apparently related to the C1/2
of the apoptosis induced by hyperthermia. The higher is
the hydrophobicity of antioxidant, the higher is the effi-
ciency against apoptosis induced by hyperthermia. On the
other hand, there was no correlation noted between the
partition coefficients and the C1/2 values in the apoptosis
induced by X-rays (Figure 4A and B).
Discussion
In this study factors involved in the anti-apoptotic activity
of a series of antioxidants against X-ray- or hyperthermia-
induced apoptosis were investigated.
760 Apoptosis ·Vol 9 ·No6·2004
Antioxidant and apoptosis
Figure 3. Relationship between rate constants of antioxidants for
O·−
2and C1/2 for apoptosis induced by X-ray or hyperthermia.
U937 cells were irradiated at doses of 10 Gy (A) or treated with
44.0◦Cfor 30 min (B) in the presence or absence of the antiox-
idants; the cells were harvested 6 hr after the treatment and the
rates of the DNA fragmentation were measured. The rate con-
stants of reaction for O ·−
2were evaluated. Values above 14 mM
along Y-axis were not shown in the figure. The data are the aver-
ages of four independent experiments.
X-irradiation is able to generate ·OH radicals. Since
·OH has extremely high rate constants for various
biomolecules, this radical rapidly react with base and
sugar moiety to cause DNA damage, and with lipid to
induce membrane lipid peroxidation. These are possible
triggers of radiation-induced apoptosis.5,6Another radi-
cal O ·−
2is known to be generated in hyperthermia that
contributes to inducing of apoptosis.9,10 In the present
study the result show that no correlation between the
ROS scavenging activity of the antioxidants (based on the
reaction constants of the agents) and its anti-apoptotic
effect (indicated by the C1/2 values). This finding sug-
gests that factors other than the ROS scavenging abil-
ity of these agents are involved in their anti-apoptotic
activity.
Figure 4. Relationship between partition coefficient in 1-
octanol/water of the antioxidants and C1/2 for apoptosis induced
by X-ray or hyperthermia. U937 cells were irradiated at doses of
10 Gy (A) or treated with 44.0◦Cfor 30 min (B) in the presence
or absence of the antioxidants; the cells were harvested 6 hr af-
ter the treatment and the rates of the DNA fragmentation were
measured. The partition coefficient in 1-octanol/water of the an-
tioxidants was evaluated. Values above 14 mM along Y-axis were
not shown in the figure. The data are the averages of four inde-
pendent experiments.
The hydrophobicity of the antioxidants correlated well
with C1/2 on hyperthermia-induced apoptosis. The data
showed that antioxidants with strong hydrophobicity
have high inhibitory effects on the apoptosis induced by
hyperthermia. This result is consistent with the known
fact that the cell membrane is an important site by
which antioxidant should interact to effect its action.
The cell membrane is composed of a lipid bilayer and
in hyperthermia-induced apoptosis, ROS target the cell
membrane to increase lipid peroxidation. This form of
damage to the membrane plays a pivotal role in induc-
ing apoptosis.10,17,25 Therefore, preventing lipid peroxi-
dation is an important factor in protecting cells against
hyperthermia-induced apoptosis. In addition, the mem-
brane is a highly selective filter serving as a barrier to
most water soluble molecules, but hydrophobic molecules
Apoptosis ·Vol 9 ·No6·2004 761
Z.-G. Cui et al.
easily interact and accumulate on the cell membrane.26
The more hydrophobic an antioxidant is, the easier it can
interact and accumulate on the cell membrane to prevent
lipid peroxidation; and can easily penetrate into cells to
scavenge the ROS.
However, in the irradiated cells, there is no correlation
between C1/2 and the hydrophobicity of antioxidants.
The lack of correlation indicates that the mechanism in-
volved in apoptosis induced by X-ray is different from
that of hyperthermia. It is known that in X-irradiated
cells ·OH are generated directly and transiently within
the cells. Therefore, multiple sites including DNA as
well as the cell membrane should be considered as tar-
gets of triggering signals for radiation-induced apoptosis.
In contrast, hyperthermia generates O ·−
2indirectly, het-
erogeneously and chronically within the cells. The het-
erogeneous O ·−
2generation most likely from mitochon-
dria and/or the heterogeneous reactive sites of O ·−
2may
explain the suppressive effects on hyperthermia-induced
apoptosis by these agents depending on their hydropho-
bicity. This lack of correlation between the partition coef-
ficient and C1/2 in radiation-induced apoptosis, also sug-
gest that hydrophobicity may not be equally an important
factor for the anti-apoptotic activity of these agents.
In conclusion, the activity of the antioxidants against
the apoptosis induced by X-rays or hyperthermia can not
be explained by their ROS scavenging ability. The hy-
drophobicity of the antioxidants is involved in their ac-
tivity against the hyperthermia-induced apoptosis, but
not in the radiation-induced apoptosis.
Acknowledgments
The authors acknowledge helpful discussions with Dr. P.
Riesz, Radiation Biology Branch, National Cancer Insti-
tute, National Institutes of Health, Bethesda, MD, USA.
This study was supported in part by a Grant-in Aid
for Scientific Research on Priority Areas (12217049), a
Grant-in Aid for Scientific Research (C) (14580565) and
a Grant-in Aid for COE Research from the Ministry of Ed-
ucation, Culture, Sports, Sciences and Technology, Japan.
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