J Pharmacol Sci 92, 166 – 170 (2003) Journal of Pharmacological Sciences

©2003 The Japanese Pharmacological Society

Short Communication

Effects of Naringin on Hydrogen Peroxide-Induced Cytotoxicity

and Apoptosis in P388 Cells
Syu-ichi Kanno*, Ai Shouji, Keiko Asou, and Masaaki Ishikawa
Department of Pharmacology and Toxicology, Cancer Research Institute, Tohoku Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan

Received December 13, 2002; Accepted April 12, 2003

Abstract. Flavonoids are widely recognized as naturally occurring antioxidants. Naringin (NG)
is one of the flavonoid components in citrus fruits such as grapefruit. Hydrogen peroxide (H2O2)
causes cytotoxicity through oxidative stress and apoptosis. In this paper, we examined the effects
of NG on H2O2-induced cytotoxicity and apoptosis in mouse leukemia P388 cells. Cytotoxicity
was determined by mitochondrial activity (MTT assay). Apoptosis and DNA damage were
analyzed by measuring chromatin condensation and Comet assay (alkaline single cell gel electro-
phoresis), respectively. H2O2-induced cytotoxicity was significantly attenuated by NG or the
reduced form of glutathione (GSH), a typical intracellular antioxidant. NG suppressed chromatin
condensation and DNA damage induced by H2O2. These results indicate that NG from natural
products is a useful drug having antioxidant and anti-apoptopic properties.

Keywords: naringin, hydogen peroxide, apoptosis

Many physiological and environmental toxicants and
chemicals (including metabolic poisons and chemo-
therapeutic drugs) are injurious to cells via oxidative
stress involving the overproduction of reactive oxygen
species (ROS), such as hydrogen peroxide (H2O2),
superoxide anions (O2•), and hydroxyl radicals (HO•).
The most valuable exogenous ROS generator is H2O2,
although it has not the properties of radical for itself.
In particular, H2O2 is a potential source for HO•, one
of the most dangerous radicals HO•, through the
Fenton reaction in the presence of transition metal ions.
Furthermore, oxidative stress causes apoptosis or DNA
damage (genotoxicity). Therefore, H2O2 can serve as a
typical chemical for investigating of oxidative stress
and apoptosis (1).
Recently, it has been suggested that oxidative stress
causes apoptosis in various diseases, such as Parkinson’s
disease (2). The oxidative stress-induced apoptosis is
attributed to not only disease but also pharmacological
action or a secondary effect of several drugs. Anti-
cancer drugs, for instance, such as doxorubicin, cytosine
arabinoside (Ara-C) and etoposide, show DNA damage
through oxidative stress either directly or by metabolic
*Corresponding author. FAX: +81-22-275-2013
E-mail: syu-kan@tohoku-pharm.ac.jp
activation, which is reduced by antioxidants. Anti-
oxidants are desirable in that they can be taken easily
using natural products.
In many natural products, bioflavonoids have been
reported to exhibit antioxidant activities (3, 4) and
inhibit lipid peroxidation in biological membranes (5).
The flavonoids having these antioxidant activities are
known to have at least one free aromatic hydroxyl group.
Previously, naringin (NG) has been demonstrated to
have antiviral (6) and antiallergic (7) actions through
the regulation of ROS. In this study, we examined the
effect of NG on H2O2-induced cytotoxicity, apoptosis,
and genotoxicity in mouse P388 cells.
Mouse leukemic P388 cells were supplied by the
Cell Resource Center for Biomedical Research, Tohoku
University (Sendai). Cells were routinely kept in RPMI
1640 medium supplemented with 10% fetal bovine
serum and penicillin G (100 U / ml) / streptomycin
(100 mg / ml) at 37°C in a humidified 5% CO2 – 95% air
incubator. Cell viability was measured by counting the
cells without staining with 0.2% trypan blue. To main-
tain exponential growth, cells were seeded at 1 ´
105 cells / ml and passaged every 4 to 5 days.
Drugs used in these experiments were as follows:
H2O2 was purchased as a 30% stock solution (Santoku

166
 Effects of Naringin on H2O2 Cytotoxicity
Chemical Industries Co., Ltd., Tokyo); NG and all other
reagents were supplied by either Sigma (St. Louis, MO,
USA) or Nacalai Tesque (Kyoto). All cell culture
reagents were obtained from Invitrogen Corp (Carlsbad,
CA, USA). NG was dissolved in dimethylsulfoxide
(DMSO). DMSO at concentrations lower than 0.5%
had no effect on cell growth. Light exposure was kept
to a minimum for all drugs used.
The P388 cells were cultured at 4 ´ 104 cells / 100 ml
per well in a 96-well plate for cytotoxicity assay or at
8 ´ 105 cells / 2 ml per well in a 24-well plate for other
assay. Cytotoxicity was determined by the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
assay with a modification of our previously method (8).
Following treatment to P388 cells with H2O2 and / or
other drugs, 10 m l of MTT (5 mg / ml saline) was added
to each well, and then the samples were incubated for
90 min at 37°C and centrifugated (300 ´ g for 5 min).
After aspiration of supernatant, the cells were lysed
and solubilized by the addition of 100 m l 0.04 N HCl
containing isopropanol. The absorbance of each sample
was analyzed at 590 nm using the Inter-med model NJ-
2300 Microplate Reader. Cytotoxicity (%) was calcu-
lated relatively to the control.
To analyze the changes in nuclear morphology, cells
were collected by centrifugation and washed with PBS.
The cells were stained for 10 min at room temperature
in PBS containing 5 m M bisbenzimide H 33342 fluoro-
chrome trihydrochloride (H 33342) and examined by
fluorescence microscopy. Apoptosis was characterized
by chromatin condensation followed by partition into
multiple bodies.
DNA damage was also detected by the Comet assay
(alkaline single cell gel electrophoresis), developed by
Tsuda et al. (9). P388 cells were treated with drugs for
1 h. Seventy-five microliters of 0.75% regular agarose
(GP-42) was quickly layered on a fully frosted slide and
covered with another slide. The cells were suspended in
0.75% low melting point agarose at 37°C, and 75 ml of
the cell suspension was quickly layered on the GP-42
layer after removal of the top slide. Finally, 75 m l of
0.75% agarose GP-42 was layered quickly over the cells.
The slides were placed immediately in a chilled lysing
solution (pH 10) of 2.5 M NaCl, 100 mM Na2EDTA,
10 mM Tris, 1% sarkosyl, 10% DMSO, and 1%
Triton X-100 and kept at 0°C in the dark for 60 min. The
slides were placed on a horizontal gel electrophoresis
platform and covered with chilled alkaline solution
made up of 0.3 M NaOH and 1 mM Na2EDTA (pH 13).
The slides were left in the solution for 20 min in the
dark at 0°C to allow DNA unwinding and expression
of alkali-labile sites. The power supply was set at 25 V
(1.0 / cm). The DNA was electrophoresed for 15 min
167
with a current of approximately 250 mA at 0°C in the
dark, and the slides were rinsed gently 2 times with
0.4 M Tris (pH 7.5) to neutralize the excess alkali.
Each slide was stained with 50 m l ethidium bromide and
covered with a coverslip. Fifty cells on one slide were
examined in a fluorescence microscope (at 200 ´ magni-
fication; Nikon, Tokyo) equipped with an excitation
filter of 515 – 560 nm and a barrier filter of 590 nm. The
whole comet length and the diameter of the head were
measured.
Statistical analysis of the results was performed with
one-way analysis of variance (ANOVA) followed by
Scheffe’s F test. P-value of less than 0.01 was consid-
ered significant.
First, we examined the concentration-dependency for
NG (0.03, 0.1, 0.3, and 1 mM) in reduction of 30 mM
H2O2-induced cytotoxicity of P388 cells (Fig. 1A). The
cells were preincubated for 15 min with various concen-
trations of NG, and then they were incubated for 24 h
with 30 mM H2O2. The 30 m M H2O2-induced cyto-
toxicity (52.3 ± 11.4%) was blocked by NG concentra-
tion-dependently. NG at the concentration of 1 mM
most significantly blocked H2O2-induced cytotoxicity
(93.6 ± 6.3%). Therefore, we selected this concentration
of NG (1 mM) for subsequent experiments. Secondly,
we examined the effects of NG and gluthathione on
H2O2-induced cytotoxicity. Treatment of P388 cells with
H2O2 alone (Vehicle) for 24 h resulted in concentration-
dependent induction of cytotoxicity. H2O2-induced cyto-
toxicity was significantly ameliorated by NG and
glutathione (GSH: 1 mM), a typical intracellular anti-
oxidant (Fig. 1B). The toxicity of H2O2 may largely
depend on the intracellular level of reduced GSH. We
examined the effects of NG and GSH on H2O2-induced
cytotoxicity in the cells pretreated with DL-buthionine-
[S,R]-sulfoximine (BSO), a specific inhibitor of g-
glutamylcysteine synthetase, to decrease cellular GSH.
BSO decreased the GSH content in the cells from
35.8 ± 2.1 nmol / mg protein to 9.7 ± 1.6 nmol / mg pro-
tein after 24 h. Although cells depleted of GSH were
viable and able to undergo at least two rounds of dupli-
cation, depletion of GSH by prolonged incubation with
BSO markedly increased the H2O2-induced cytotoxicity
(Fig. 1C). The treatment with H2O2 after the incubation
with BSO resulted in the increase in cytotoxicity by 9 –
10-fold over the respective control (ED50 values for
control and BSO pretreated cells were 32.5 ± 6.3 mM
and 3.5 ± 0.4 m M, respectively). Nevertheless, NG or
GSH significantly attenuated the H2O2-induced cyto-
toxicity. GSH is the most abundant intracellular anti-
oxidant in mammals and plays an important role in
various cellular functions. It has been reported that
extracellular supplementation of GSH in culture medium
168 S Kanno et al
Fig. 1. The protection effect by naringin (NG) on H2O2-induced
cytotoxicity. Dose-dependent effect of NG on 30 m M H2O2-induced
cytotoxicity (A). The effect of 1 mM NG or 1 mM GSH on H2O2-
induced cytotoxicity in control cells (B) or DL-buthionine-[S,R]-
sulfoximine (BSO)-pretreated cells (C). P388 cells were incubated
with H2O2 in the presence or absence of NG or GSH at 37°C for
24 h. Cells pretreated with BSO at 1 mM were exposed to H2O2 for
24 h. Cytotoxicity was determined by the MTT assay as described in
Materials and Methods. Each column or point represents the mean
value ± S.E.M. (n = 5). *P<0.01, significantly different from the
cells exposed to H2O2 alone (vehicle).
significantly abolished all forms of oxidant-mediated
cell death (10) and provided useful radical scavengers
for the hydroxy radical (11). It is thought that the anti-
oxidant activity of NG is similar to that of GSH. Further-
more, H2O2-induced cell lipid peroxidation was blocked
by NG (data not shown).
Next, we examined the effect of NG on H2O2-induced
apoptosis. H2O2 has been reported to trigger apoptosis
in various cell types (1, 10). We detected apoptosis by
changes in nuclear morphology with DNA staining. The
DNA staining with H 33342 dyes in P388 cells revealed
different nuclear features of cells treated with H2O2
(Fig. 2). The nucleus of P388 treated with 30 mM H2O2
had features consistent with those of apoptosis (i.e., cell
shrinkage, nuclear condensation, and fragmentation)
(Fig. 2A). H2O2-induced apoptosis was decreased by
NG at each concentration of H2O2 (Fig. 2B), although
high concentration of H2O2 (100 m M)-induced apoptosis
was almost unaffected by NG. Thus, we presumed that
the protective effect of NG on H2O2-induced cyto-
toxicity was caused by potent inhibition of H2O2-
induced apoptosis.
The Comet assay (alkaline single-cell gel electro-
phoresis) has been used as a test system to detect DNA
damage. The Comet assay has several advantages in-
cluding its simple and rapid performance, its sensitivity
for detecting a broad spectrum of DNA damage, the
analysis of data at the level of the individual cell, the
use of extremely small samples, and its applicability to
virtually any karyote cell population (12). The Comet
assay is a highly sensitive way to detect DNA damage
induced by oxidative stress, because H2O2-induced
genotoxicity can be detected before producing cell death
(13). Then, we examined the effect of NG on H2O2-
induced DNA damage for 1 h, using the Comet assay.
A typical image is shown in Fig. 3A. When there is no
DNA damage, the cell shows no or a few tails. When
there is DNA damage in the cells, many cells had tails
like comets. The DNA migration was calculated as the
index of DNA damage, and the results obtained after
H2O2 treatment with or without NG are shown in
Fig. 3B. NG completely blocked the DNA migration
induced by 10 m M and 30 mM H2O2 but not that by
100 m M H2O2. Apoptotic cells, manifested as Comets
having well-separated heads and tails, were detected in
this experiment. H2O2 produced extensive DNA damage
after 1 h even in the absence of cytotoxicity. Thus, we
believe that H2O2 is primarily responsible for the DNA
damage in this cell line and that the cytotoxicity arose
secondarily from DNA damage.
Recently, NG has been demonstrated to play an
important role in regulating antioxidative capacity by
increasing superoxide dismutase (SOD) and catalase
 Effects of Naringin on H2O2 Cytotoxicity 169

Fig. 2. Effect of NG on H2O2-induced apoptosis at 24 h. H2O2-induced apoptosis was detected by fluorescence microscopy.
The cells were stained with 5 mM H 33342. NG was added 15 min before incubation with H2O2. A: Nuclear morphological
change in 100 m M H2O2-induced apoptosis detected by fluorescence microscopy. Magnification: ´400. B: Quantitative analysis
of H2O2-induced apoptosis by NG. *P<0.01, significantly different from the cells exposed to vehicle at the same time.

Fig. 3. DNA damage (genotoxicity) detected in P388 cells 1 h after treatment with different concentrations of H2O2 in the
presence or absence of NG. H2O2-induced genotoxicity was detected by the Comet assay (alkaline single cell gel electrophoresis)
as described in Materials and Methods. NG was added 15 min before incubation with H2O2. A: A Typical image of the comets
(100 mM H2O2). B: The quantitative results for DNA migration. Fifty cells on one slide were examined to measure the length of
the whole comet and the diameter of the head. Each column represents the mean value ± S.E.M. (n = 5). *P<0.01, significantly
different from the cells exposed to vehicle.

activities and by up-regulating the gene expression of
SOD, catalase, and glutathione peroxidase (GSH-Px) in
rabbits fed a cholesterol-rich diet (14). Since NG
blocked H2O2-induced cytotoxicity and apoptosis in the
present study, we speculate that NG might affect H2O2-
induced expression of an apoptosis-associated gene or
protein as one of its pharmacological actions. We have
to examine this point in the future.
In conclusion, H2O2-induced cytotoxicity, apoptosis
and genotoxicity were significantly prevented by NG.
NG is a main component of bioflavonoids in grapefruit
so that one can simply take it as food. The present results
suggest that NG is a useful drug having antioxidant and
anti-apoptopic activity as a natural product.
170 S Kanno et al
Acknowledgments
This study was in part supported by the Laboratory of
Veterinary Public Health, Department of Veterinary
Medicine, Faculty of Agriculture, Iwate University. The
authors are grateful to Dr. Shuji Tsuda for his critical
reading of the manuscript.
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