Safrole oxide induces neuronal apoptosis through inhibition of integrin β4/SOD activity and elevation of ROS/NADPH oxidase activity

Article abstract of DOI:10.1016/j.lfs.2006.11.041

Neuronal apoptosis is a very important event in the development of the central nervous system (CNS), but the underlying mechanisms remain to be elucidated. We have previously shown that safrole oxide, a small molecule, induces integrin β4 expression and promotes apoptosis in vascular endothelial cells. In this study, the effects of safrole oxide on cell growth and apoptosis have been examined in primary cultures of mouse neurons. Safrole oxide was found to significantly inhibit neuronal cell growth and to induce apoptosis. The inhibitory and apoptotic activities of safrole oxide followed a dose- and time-dependent manner. Interestingly, the expression of integrin β4 was significantly inhibited with safrole oxide treatment. Furthermore, safrole oxide dramatically increases the level of intracellular reactive oxygen species (ROS) and the activity of NADPH oxidase. Moreover, manganese-dependent superoxide dismutase (MnSOD) activity was decreased significantly with safrole oxide treatment. Our study thus demonstrates that safrole oxide induces neuronal apoptosis through integrin β4, ROS, NADPH, and MnSOD.

Full text of DOI:10.1016/j.lfs.2006.11.041

Life Sciences 80 (2007) 999–1006
www.elsevier.com/locate/lifescie
Safrole oxide induces neuronal apoptosis through inhibition of integrin
β4/SOD activity and elevation of ROS/NADPH oxidase activity
Le Su a,c, BaoXiang Zhao b, , Xin Lv a,c, Nan Wang a,c, Jing Zhao a,c
,
⁎
ShangLi Zhang a,c, JunYing Miao a,c,
⁎
a
Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
b
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
c
The Key Laboratory of Experimental Teratology, Ministry of Education, Jinan, 250012, China
Received 13 July 2006; accepted 21 November 2006
Abstract
Neuronal apoptosis is a very important event in the development of the central nervous system (CNS), but the underlying mechanisms remain
to be elucidated. We have previously shown that safrole oxide, a small molecule, induces integrin β4 expression and promotes apoptosis in
vascular endothelial cells. In this study, the effects of safrole oxide on cell growth and apoptosis have been examined in primary cultures of mouse
neurons. Safrole oxide was found to significantly inhibit neuronal cell growth and to induce apoptosis. The inhibitory and apoptotic activities of
safrole oxide followed a dose- and time-dependent manner. Interestingly, the expression of integrin β4 was significantly inhibited with safrole
oxide treatment. Furthermore, safrole oxide dramatically increases the level of intracellular reactive oxygen species (ROS) and the activity of
NADPH oxidase. Moreover, manganese-dependent superoxide dismutase (MnSOD) activity was decreased significantly with safrole oxide
treatment. Our study thus demonstrates that safrole oxide induces neuronal apoptosis through integrin β4, ROS, NADPH, and MnSOD.
© 2006 Published by Elsevier Inc.
Keywords: Neurons; Apoptosis; Integrin β4; Reactive oxygen species; Safrole oxide
Introduction
Du et al., 2005, 2006; Su et al., 2006). However, the specific
cellular and molecular mechanisms underlying safrole oxide's
effect on cell survival and cell apoptosis still need to be
elucidated in various cells.
Safrole oxide has been studied for many years (Noller and
Kneeland, 1964). Previous studies have shown that complexes
of safrole oxide and DNA were formed in vitro between safrole
oxide and deoxyguanosine (Qato and Guenthner, 1995).
Furthermore, recent studies have indicated that safrole oxide
could form covalent bonds with DNA in vitro, binding
primarily to DNA bases. Epoxide hydrolases can prevent the
binding of safrole oxide to DNA (Luo and Guenthner, 1996).
We recently reported that safrole oxide induces apoptosis in
various cell types, including vascular endothelial cells and
human lung cancer cells (Miao et al., 2002; Zhao et al., 2005;
Integrins, a large family of heterodimeric receptors, which
consist of α and β subunits, play fundamental roles in cellular
interactions and motility during mammalian development
(Tarone et al., 2000). Integrin β4, an exceptionally large cyto-
plasmic domain of integrin family, consists of more than 1000
amino acids and associates with cytoskeletal and signaling
molecules (Hogervorst et al., 1990; Tamura et al., 1990). Recent
reports indicate that integrin β4 plays an important role in
cellular signal transduction pathways, such as nuclear factor-
kappaB (NF-κB) pathway (Sakakura et al., 2002; Weaver et al.,
2002). Integrin β4 has important functions in the nervous
system. It has been implicated in the initiation of myelin sheath
formation in Schwann cells (Einheber et al., 1993; Feltri et al.,
1994; Niessen et al., 1994). Strikingly, increasing evidence
indicated that integrin β4 has cell-type-specific functions
⁎
Corresponding authors. Institute of Developmental Biology, School of Life
Science, Shandong University, Jinan 250100, China. Tel.: +86 531 88364929;
fax: +86 531 88565610.
E-mail addresses: bxzhao@sdu.edu.cn (B. Zhao), miaojy@sdu.edu.cn
(J. Miao).
0024-3205/$ - see front matter © 2006 Published by Elsevier Inc.
doi:10.1016/j.lfs.2006.11.041

1000
L. Su et al. / Life Sciences 80 (2007) 999–1006
(Jacques et al., 1998; Whitley et al., 2005; Zhang and Galileo,
1998). However, the roles of integrin β4 in neuronal survival
and apoptosis remain unknown.
Cell viability analysis
Cell viability was determined by MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltertrazolium bromide) assay (Price and McMil-
lan, 1990). Briefly, cells were seeded into 96-well plates and
treated with or without safrole oxide. After treatments as indicated,
the cells were incubated with 0.5% MTT for 4 h. The medium was
removed and 100 μl of 0.04 M dimethylsulfoxide solution was
added. The absorbance of the reaction product in solution at
570 nm was measured using a SpectraMAX 190 microplate
spectrophotometer (GMI Co., USA). The percentage of living
cells was calculated by the ratio of OD. The morphological
changes of the neurons cultured in 24-well cell culture plates were
observed with a phase contrast microscope (Nikon, Japan).
Our previous studies have shown that safrole oxide induces
vascular endothelial cell (VEC) apoptosis through up-regulation
of integrin β4 and inhibition of intracellular reactive oxygen
species (ROS) (Zhao et al., 2005). Although apoptosis plays a
vital role in neuronal diseases, the molecular mechanisms of
neuronal survival and apoptosis are not well understood. We
investigated the mechanisms by which safrole oxide induces
mouse neurons. Specifically we determined the involvement of
the integrin β4/superoxide dismutase (SOD), and ROS/
NADPH oxidase activity. We show in this study that safrole
oxide promotes neuronal apoptosis by the modulation of
integrin β4/SOD activity and via elevation of ROS/NADPH
oxidase activity.
Analysis of nuclear condensation and fragmentation by
acridine orange staining
Materials and methods
After treatment with 70 μg/ml safrole oxide for 20 h, the cells
were stained with 5 μg/ml acridine orange for 5 min at room
temperature. The nuclear condensation and fragmentation were
observed with a laser scanning confocal microscope (Leica,
Germany).
Cell culture and identification
Mouse neurons were obtained from embryo forebrains at
the 14th day of gestation under the sterile condition (Sunanda
et al., 1998). The cortexes were cut into small pieces about
1.0 mm3 in size. After trypsinization (0.125%) for 20 min at
37 °C, Dulbecco's modified Eagle's medium (DMEM)
(Gibco, USA) supplemented with 10% fetal bovine serum
(FBS) (Hyclone, USA) was added to the cell suspension to
end this digestion. Then the cells were seeded at a density of
4×105 cells/cm2 in poly-L-lysine-coated plastic dishes. The
supplemented culture medium consisted of DMEM with
10% FBS, 0.1 mM L-glutamine and 4 mM D-glucose. Cells
were grown at 37 °C in 5% CO2 and 95% air with saturated
humidity.
To identify mouse neurons, we determined the expressions
of neuron-specific enolase (NSE), neurofilament-L (NF-L),
synapsin and glial fibrillary acidic protein (GFAP) using rabbit
anti-mouse IgG (Santa Cruz, USA, 1:100 dilution) and
immunofluorescence staining as described by Wang et al.
(2004). Samples were evaluated under laser scanning confocal
microscope (Leica, Germany).
LDH assay
Lactate dehydrogenase (LDH) assay was performed on cells
treated with 70 μg/ml safrole oxide for 20 h using a LDH kit
(ZhongSheng, China) according to the manufacturer's protocol.
Light absorption was measured at 340 nm using a model Cintra
5 UV–vis spectrometer (GBC, Australia). LDH activity was
calculated by the following formula:
LDH ðU=LÞ ¼ ð⊿Asample=min−⊿Ablank=minÞ ꢀ F
F ¼ 1000 ꢀ Vtotal=ðVsample ꢀ extinction coefficientÞ
The extinction coefficient of NADH in mmol at 340 nm was 6.3.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end
labeling (TUNEL) assay
Exposure to safrole oxide
The TdT-mediated dUTP nick-end labeling technique was
used to detect in situ nuclear DNA fragmentation and measure the
apoptosis ratio (Gavrieli et al., 1992). In brief, after cells were
treated in the presence or absence of 70 μg/ml safrole oxide for
20 h, DNA fragmentation was detected by the DeadEndTM
Fluorometric TUNEL System (Promega, USA) according to the
manufacturer's protocol. Cells were evaluated by a laser scanning
confocal microscope (Leica, Germany). The percent apoptosis
rate was quantified according to the TUNEL-positive rate.
Safrole oxide [3,4-(methylenedioxy)-1-(2′,3′-epoxypropyl)-
benzene] was synthesized by treating safrole with 3-chloroper-
oxybenzoic acid and purified by silica gel column chromatog-
raphy (Zhao et al., 2003). It was dissolved in ethanol and
applied to cells such that the final concentration of ethanol in the
culture medium was below 0.01%. Ethanol at a concentration of
0.1% did not affect the viability of the cells. The cells were
divided into two groups: in normal group, cells were cultured in
the supplemented medium; in the safrole oxide treatment group,
the cells were incubated with 10, 40, 70, or 100 μg/ml safrole
oxide for 10, 20 or 30 h in the supplemented medium. The
morphological changes of the cells were observed with a phase
contrast microscope (Nikon, Japan).
Immunofluorescence assay
Expression of integrin β4 was examined using an immuno-
fluorescence assay. After treatments, the cells were washed

L. Su et al. / Life Sciences 80 (2007) 999–1006
1001
Fig. 1. Identification of primary culture neurons. Mouse neurons after being isolated for 3 days were cultured in the supplemented medium and expressions of NSE,
NF-L, synapsin, and GFAP were determined using rabbit anti-mouse IgG and immunofluorescence staining as described under Materials and methods. Data presented
are representative of three independent experiments.
three times with 0.1 M PBS. Following fixation (15 min in 4%
paraformaldehyde), the cells were washed three times in 0.1 M
PBS and blocked with sheep serum (1:50 dilution) in 0.1 M
PBS for 20 min at room temperature. Then cells were stained
with rabbit anti-mouse integrin β4 IgG (Santa Cruz, USA,
1:100 dilution) in a humid chamber overnight at 4 °C. The cells
were washed three times with 0.1 M PBS and incubated for
30 min at 37 °C with the secondary antibodies, FITC-
conjugated goat anti-rabbit (Santa Cruz, USA, 1: 200 dilution).
Finally the cells were washed three times with 0.1 M PBS. Then
the samples were evaluated by a laser scanning confocal
microscope (Leica, Germany). We randomly selected the region
of interest (ROI), then zoomed in the same frames. The value of
relative fluorescent intensity per cell equals the total value of
sample in scan zoom divided by the total number of cells (at
least 200 cells) in same zoom.
(Nanjing Jiancheng, China). The optical density was measured
at 550 nm (wavelength). The enzyme activity was expressed as
U/mg of protein.
Cytochrome c reduction assay
NADPH oxidase activity (NADPH-dependent O2− produc-
tion) in cell homogenates was examined using the SOD-
inhibitable cytochrome c reduction assay (Li et al., 2002). Cell
homogenate (final concentration 1 mg/ml) diluted in DMEM
Intracellular ROS assay
The levels of intracellular ROS were detected in cells treated
with or without 70 μg/ml safrole oxide using a fluorescent
probe, 2′,7′-dichlorodihydrofluorescin (DCHF, Sigma, USA),
which can be oxidized into fluorescent 2′,7′-dichlorofluorescin
(DCF) by intracellular ROS. This assay is a reliable method for
measuring the intracellular ROS (Suematsu et al., 2003). The
fluorescence was monitored with laser scanning confocal
microscopy (Leica, Germany). The amount of ROS was
quantified as the relative fluorescence intensity of DCF per
cell in the scanned area.
SOD activity assay
The neurons were treated in the presence or absence of
70 μg/ml safrole oxide for 20 h. Then the enzyme samples were
prepared as described by Wu et al. (1997). Briefly, the cells were
washed twice with PBS and trypsinized. They were homoge-
nized with ultrasonic treatment (400 W, 5 min) in 500 μl
physiological salt solution on ice. After centrifugation at
10,000×g, 4 °C for 10 min, the supernatant was used for the
SOD activity assay. The levels of SOD in cells were assessed
according to the instructions provided by SOD detection kit
Fig. 2. Effects of safrole oxide on the morphology and cell viability of primary
cultures of neurons. (a) We cultured mouse neurons after being isolated for
3 days in the supplemented medium and treated with or without safrole oxide at
70 μg/ml for 20 h. The morphological changes were examined by a phase
contrast microscope (×400). Data presented are representative of three
independent experiments. (b) Mouse neurons after being isolated for 3 days
were treated with or without safrole oxide at various concentrations for different
periods. The viability of neurons was determined by MTT assay as described
⁎
⁎⁎
under Materials and methods. Pb0.05, Pb0.01 vs. normal. Mean values
were derived from three independent experiments.

1002
L. Su et al. / Life Sciences 80 (2007) 999–1006
without phenol red was distributed in 96-well flat-bottomed
culture plates (final volume 200 μl/well). Cytochrome c
(500 μmol/l) and NADPH (100 μmol/l) were added in the
presence or absence of SOD (200 U/ml) and incubated at room
temperature for 30 min. Cytochrome c reduction was measured
by reading the absorbance at 550 nm on a microplate reader. O2−
production in nmol/mg protein was calculated from the
difference between absorbance with or without SOD and the
extinction coefficient for the change of ferricytochrome c to
Safrole oxide significantly inhibits growth of neuronal cells
We have reported that safrole oxide induces apoptosis in
various cell types (Miao et al., 2002; Su et al., 2006). Apoptotic
cell death has an important role in neuronal diseases. However,
the role of safrole oxide in neuronal cell death and apoptosis
remains to be defined. To determine whether safrole oxide
induces neuronal cell death and apoptosis, we treated mouse
neurons with or without safrole oxide at 10, 40, 70 and 100 μg/
ml. At 10, 20, and 30 h later, cell viability was determined by
MTT assay. We found that the inhibitory effect of safrole oxide
was dose- and time-dependent, and the MTT assay was reduced
to about 31% of the control level by safrole oxide at 70 μg/ml
for 30 h (Fig. 2b). Safrole oxide treatment at 70 μg/ml for 20 h
was used in the subsequent experiments. Morphological effect
of safrole oxide was further tested in neuronal cells as shown in
Fig. 2a. Safrole oxide effectively induced neuronal cell death
and apoptotic bodies, as assessed using a phase contrast
microscope.
ferrocytochrome c, i.e., 21.0 mmol l−1 cm−1
.
Statistical analysis
Data were expressed as mean S.E. and accompanied by the
number of independent experiments. Statistical analysis was
done by t test, and differences of Pb0.05 were considered
statistically significant.
Results
The identification of neurons
Safrole oxide induces apoptosis in neuronal cells
To identify mouse neurons, we examined the expressions of
specific markers for neurons by immunofluorescence staining,
including NSE, NF-L and synapsin (Wang et al., 2006). Our
data showed more than 98% neuronal cells (Fig. 1). We also
showed negative control for GFAP staining (Fig. 1).
We next examined whether safrole oxide could induce
neuronal cell necrosis, an extreme end of neurotoxicity. Results
from LDH assays showed that there was no significant
difference in LDH release in neuronal cells between the normal
group and the treatment group with safrole oxide at 70 μg/ml
Fig. 3. Safrole oxide triggered neurons to undergo apoptosis. (a) Mouse neurons after being isolated for 3 days were cultured under the supplemented medium in the
presence or the absence of safrole oxide (70 μg/ml). After a 20-h treatment, LDH assay was performed using a LDH kit as described under Materials and methods.
&PN0.05 compared normal group. Mean values were derived from three experiments. (b) We treated mouse neurons after being isolated for 3 days with or without
70 μg/ml safrole oxide. 20 h later, the cells were stained with acridine orange for 5 min and then determined nuclear fragmentation by fluorescent microscopy. Data
presented are representative of three independent experiments. (c) Mouse neurons after being isolated for 3 days were cultured under supplemented medium with or
without safrole oxide at 70 μg/ml for 20 h. Both nuclear condensation and fragmentation were then detected by TUNEL assay as described under Materials and
methods. Data presented are representative of three independent experiments. (d) The quantity of apoptotic cells. The neurons were treated with or without 70 μg/ml
safrole oxide. 20 h later, apoptotic cells were determined by TUNEL assay and the percent apoptosis was calculated as described under Materials and methods.
⁎⁎
Pb0.01 compared to normal. Mean values were derived from three experiments.

L. Su et al. / Life Sciences 80 (2007) 999–1006
1003
2002; Weaver et al., 2002). However, little is known about the
importance of integrin β4 in neurons. We have shown that
safrole oxide induces vascular endothelial cell apoptosis
through up-regulation of integrin β4 (Zhao et al., 2005). We
therefore investigated the safrole oxide effects on the expression
of integrin β4 in neurons. We and others recently reported that
immunofluorescence combined with laser scanning confocal
microscopy is a useful method for detecting protein changes.
These techniques allow semi-quantitative evaluation of protein
expression (Dinser et al., 2001; Zhao et al., 2004b). We treated
neurons with or without safrole oxide and found that the
expression of integrin β4 was markedly reduced with safrole
oxide treatment compared with the normal (Fig. 4a and b). The
negative control lacked primary antibody and demonstrated that
the staining was specific (data not shown). These results
indicate that integrin β4 plays a role in safrole oxide-induced
apoptosis in neurons.
Effects of safrole oxide on the levels of intracellular ROS
To further understand the possible mechanisms by which
safrole oxide induced apoptosis in the neurons, we measured the
levels of intracellular ROS. As shown in Fig. 5, safrole oxide
significantly increased the level of ROS in neurons. Our results
showed that safrole oxide induced neuronal apoptosis by
elevating the levels of intracellular ROS.
Fig. 4. Effects of safrole oxide on the expression of integrin β4 in primary
cultures of neurons. (a) Mouse neurons after being isolated for 3 days were
cultured under the supplemented medium in the presence or the absence of
safrole oxide (70 μg/ml). After a 20-h treatment, the expression of integrin β4
was determined using immunofluorescence assay as described under Materials
and methods. Data presented are representative of three independent experi-
ments. (b) The quantity of integrin β4 expression in the two groups mentioned
above. The value of the figure represented the relative fluorescent intensity per
⁎⁎
cell determined by laser scanning confocal microcopy. Pb0.01 compared to
normal. Data presented are representative of three independent experiments.
safrole oxide for 20 h (Fig. 3a), indicating that safrole oxide did
not promote necrosis in neurons. To determine whether safrole
oxide could induce neuronal apoptosis, neuronal cells
were treated with or without 70 μg/ml safrole oxide. After a
20-h treatment, morphological changes were detected using
acridine orange staining by fluorescent micrographs. We found
that safrole oxide induced neuronal cell nuclear condensation
and fragmentation, which are characteristics of apoptosis (Fig.
3b). Therefore, our results demonstrated that safrole oxide
promoted neuronal cell apoptosis. We further assessed neuronal
apoptosis using a TUNEL technique. We found that a
significant number of cells in the treatment with safrole oxide
were undergoing apoptosis, whereas only a few apoptotic cells
were detected in the control group (Fig. 3c). In safrole oxide-
treated cells, there were about 54% cells stained positive for
DNA fragmentation which was significantly higher than the
control about 6% (Fig. 3d). Thus, safrole oxide effectively
induced apoptosis in the neurons.
Fig. 5. Effects of safrole oxide on the level of ROS in the neurons. (a) The
relative intensity of ROS in the neurons. Mouse neurons after isolated for 3 days
were cultured in supplemented medium and in the presence or the absence of
safrole oxide (70 μg/ml). 20 h later, the relative intensity of ROS was detected
with laser scanning confocal microscopy. (b) The quantity of ROS level in the
two groups mentioned above. The value of the figure represented the relative
fluorescent intensity per cell determined by laser scanning confocal microcopy.
Safrole oxide attenuates the expression of integrin β4
Increasing evidence indicates that integrin β4 has an
important role in cellular signal transduction pathways, such
as nuclear factor-kappaB (NF-κB) pathway (Sakakura et al.,
⁎⁎
Pb0.01 compared to normal. Mean values were derived from three
experiments.

1004
L. Su et al. / Life Sciences 80 (2007) 999–1006
normal CNS development. To further understand the mechan-
isms of CNS development, we investigated the effects of safrole
oxide on the survival and apoptosis of mouse neurons from E14.
Recently, chemical genetics, which is using small molecules
to induce changes in cellular phenotypes, has had a significant
impact in diverse areas of developmental biology and cell
biology (Spring, 2005). The majority of these biologically active
compounds are effective and specific inhibitors of known
intracellular signaling pathways. Moreover, the identification of
small molecules with specific biological properties and
unknown intracellular targets can also lead to significant
insights into various biological questions (Smukste and Stock-
well, 2005). We have synthesized a series of small molecules,
including safrole oxide and its derivatives, such as isochroman
compounds, novel 3-morpholinone derivatives and others (Zhao
et al., 2003, 2004a; Zuo et al., 2004). We found that safrole
oxide induces VEC proliferation, differentiation and apoptosis
at different concentrations (Miao et al., 2002; Zhao et al., 2005;
Su et al., 2006). These results suggested that safrole oxide might
be a useful tool for investigating the mechanism of apoptosis.
It has been suggested that integrin β4 plays an important role
in cellular signal transduction pathways (Sakakura et al., 2002;
Weaver et al., 2002). Several reports have shown that integrin
β4 has important functions in the nervous system. It has been
implicated in the initiation of myelin sheath formation in
Schwann cells (Einheber et al., 1993; Feltri et al., 1994; Niessen
et al., 1994). However, the role of integrin β4 in neuron survival
and apoptosis remains unknown. We have reported that safrole
oxide could induce VEC apoptosis by affecting the expression
and the distribution of integrin β4 (Zhao et al., 2005). In this
study, we found that the expression of integrin β4 was inhibited
in the neuronal apoptosis induced by safrole oxide. Our data
suggest that integrin β4 is involved in the regulations of neuron
survival and apoptosis.
Fig. 6. Effects of safrole oxide on SOD and NADPH oxidase activities in the
neurons. (a) We treated mouse neurons after being isolated for 3 days with or
without 70 μg/ml safrole oxide. 20 h later, total activities of SOD were
determined by SOD detection kit as described under Materials and methods. The
#1
⁎⁎
activities of MnSOD and Cu–ZnSOD were also determined. Pb0.01 vs.
Pb0.05 vs.
.
.
&PN0.05 vs. #3. Mean values were derived from three
#2
⁎
experiments. (b) The activity of NADPH oxidase was examined using the SOD-
inhibitable cytochrome c reduction assay in the neurons treated with or without
#
⁎
70 μg/ml safrole oxide for 20 h. Pb0.05 vs. . Mean values were derived from
three experiments.
Effects of safrole oxide on the activities of SOD and NADPH
oxidase
Recent studies have shown that ROS act as intracellular
messengers in signal transduction pathways, and nontoxic levels
of ROS may play essential roles as signaling molecules,
regulating cell growth and differentiation (Shibata et al., 2003).
We have found that both integrin β4 and ROS were implicated
in the neuronal apoptosis induced by D609 (Lv et al., 2006). The
results of our study showed that the level of intracellular ROS
was enhanced significantly while the expression of integrin β4
was attenuated in safrole oxide-induced neuronal apoptosis. Our
data suggested that there was a close relationship between
integrin β4 and ROS in neuronal survival and apoptosis.
To examine the mechanism by which integrin β4 regulates
the intracellular levels of ROS during neuron survival and
apoptosis, we investigated the effects of integrin β4 down-
regulation induced by safrole oxide on SOD and NADPH
oxidase. Balancing the functions of ROS and the antioxidant
defense mechanism is thought to be essential for the regulation
and prevention of apoptosis. One efficient antioxidant enzyme
in neurons is SOD (Sakudo et al., 2003). This enzyme converts
superoxide anion radicals into H2O2. There are two isoforms of
SOD, including the copper/zinc-dependent isoform (Cu/
ZnSOD) localized in the cytoplasm and the manganese-
dependent isoform (MnSOD) localized in the mitochondria
To determine which enzymes participate in regulating the
levels of ROS in the apoptosis process induced by safrole oxide,
we examined the alterations in SOD and NADPH oxidase
activities. As shown in Fig. 6a, the activity of MnSOD
decreased significantly in the neurons treated with safrole
oxide (Pb0.05). However, safrole oxide treatment could not
alter the activity of Cu–ZnSOD (Fig. 6a). Interestingly, the
activity of NADPH oxidase was significantly increased with
safrole oxide treatment compared to the normal group (Fig. 6b).
These results indicate that both MnSOD and NADPH oxidase
were implicated in the control of ROS level during the induction
of apoptosis by safrole oxide.
Discussion
Neurons are important for maintaining the functions of the
central nervous system (CNS). Along with proliferation and
differentiation of neural cells, cell death must occur during the
development of the nervous system (Sommer and Rao, 2002).
During the proliferative stage of neurogenesis, between E13 and
E17 (Benitez et al., 2003), the regulation of neural cell survival,
apoptosis, growth and differentiation are very important for

L. Su et al. / Life Sciences 80 (2007) 999–1006
1005
Brailoiu, E., Hoard, J.L., Filipeanu, C.M., Brailoiu, G.C., Dun, S.L., Patel, S.,
Dun, N.J., 2005. Nicotinic acid adenine dinucleotide phosphate potentiates
neurite outgrowth. Journal of Biological Chemistry 280, 5646–5650.
Dickey, D.M., Aarhus, R., Walseth, T.F., Lee, H.C., 1998. Thio-NADP is not an
antagonist of NAADP. Cell Biochemistry and Biophysics 28, 63–73.
Dinser, R., Florian, K., Zaucke, F., Blank, C., Paulsson, M., Kochanek, S.,
Maurer, P., 2001. Comparison of long-term transgenic expression after non-
viral and adenoviral gene transfer into primary articular chondrocytes.
Histochemistry and Cell Biology 116, 69–77.
Du, A.Y., Zhao, B.X., Yin, D.L., Zhang, S.L., Miao, J.Y., 2005. Discovery of a
novel small molecule, 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol,
that induces apoptosis in A549 human lung cancer cells. Bioorganic and
Medicinal Chemistry 13, 4176–4183.
Du, A.Y., Zhao, B.X., Yin, D.L., Zhang, S.L., Miao, J.Y., 2006. Safrole oxide
induces apoptosis by activating caspase-3, -8, and -9 in A549 human lung
cancer cells. Bioorganic and Medicinal Chemistry Letters 16, 81–83.
Einheber, S., Milner, T.A., Giancotti, F., Salzer, J.L., 1993. Axonal regulation of
Schwann cell integrin expression suggests a role for α6β4 in myelination.
Journal of Cell Biology 123, 1223–1236.
Feltri, M.L., Scherer, S.S., Nemni, R., Kamholz, J., Vogelbacker, H., Scott, M.O.,
Canal, N., Quaranta, V., Wrabetz, L., 1994. β4 Integrin expression in
myelinating Schwann cells is polarized, developmentally regulated and
axonally dependent. Development 120, 1287–1301.
Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A., 1992. Identification of
programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. Journal of Cell Biology 119, 493–501.
Hogervorst, F., Kuikman, I., Borne, A., Sonnenberg, A., 1990. Cloning and
sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique
118 kD cytoplasmic domain. EMBO Journal 9, 756–770.
Jacques, T.S., Relvas, J.B., Nishimura, S., Pytela, R., Edwards, G.M., Streuli, C.
H., ffrench-Constant, C., 1998. Neural precursor cell chain migration and
division are regulated through different beta1 integrins. Development 125,
3167–3177.
Kuzin, B., Roberts, I., Peunova, N., Enikolopov, G., 1996. Nitric oxide regulates
cell proliferation during Drosophila development. Cell 87, 639–649.
Li, J.M., Mullen, A.M., Yun, S., Wientjes, F., Brouns, G.Y., Thrasher, A.J.,
Shah, A.M., 2002. Essential role of the NADPH oxidase subunit p47(phox)
in endothelial cell superoxide production in response to phorbol ester and
tumor necrosis factor-alpha. Circulation Research 90, 143–150.
Luo, G., Guenthner, T.M., 1996. Covalent binding to DNA in vitro of 2′,3′-
oxides derived from allylbenzene analogs. Drug Metabolism and Disposi-
tion 24, 1020–1027.
Lv, X., Wang, N., Su, L., Zhang, S.L., Miao, J.Y., 2006. Inhibition of PC-PLC
blocked the survival of mouse neural cells by up-regulating the expression of
integrin beta4 and Rb. Developmental Neuroscience 28, 499–504.
Marcus, D.L., Strafaci, J.A., Freedman, M.L., 2006. Differential neuronal
expression of manganese superoxide dismutase in Alzheimer's disease.
Medical Science Monitor 12, BR8–BR14.
Miao, J.Y., Zhao, B.X., Li, H.H., Zhang, S.L., Du, C.Q., 2002. Effect of safrole
oxide on cell growth and apoptosis induced by deprivation of growth factor
in vascular endothelial cells. Acta Pharmacologica Sinica 23, 323–326.
Niessen, C.M., Cremona, O., Daams, H., Ferraresi, S., Sonnenberg, A.,
Marchisio, P.C., 1994. Expression of the integrinα6β4 in peripheral nerves:
localization in Schwann and perineural cells and different variants of theβ4
subunit. Journal of Cell Science 107, 543–552.
Nikolova, S., Lee, Y.S., Kim, J.A., 2005. Rac1-NADPH oxidase-regulated
generation of reactive oxygen species mediates glutamate-induced apoptosis
in SH-SY5Y human neuroblastoma cells. Free Radical Research 39,
1295–1304.
Noller, R.C., Kneeland, P.D., 1964. The synthesis of some tertiary amino
alcohols related to chelidonine. Journal of the American Chemical Society
68, 201–202.
(Marcus et al., 2006). In this study, we found that the activity of
MnSOD was significantly suppressed in the neurons treated
with safrole oxide. The Cu/ZnSOD activity was not changed.
Hereby, the total SOD activity was suppressed significantly.
NADPH oxidase is another pivotal enzyme involved in the
regulation of ROS production. The NADPH oxidase is normally
dormant. But it can be rapidly activated with appropriate
stimulation and generates million molar amounts of superoxide
in a process that requires NADPH as cofactor (Tammariello
et al., 2000). It has been reported that NADPH oxidase is
unexpectedly present in neurons and can contribute to neuronal
apoptosis (Nikolova et al., 2005). In this study, we found that
the activity of NADPH oxidase was elevated significantly in the
neurons treated with safrole oxide. The data suggested that the
elevation of ROS levels might result from the increased
NADPH oxidase activity in this apoptosis process. It has been
reported that exogenous NAADP could potentiate neurite
outgrowth (Brailoiu et al., 2005). In our study, the level of
NADP+ was also elevated during the neurite degeneration
process because of the increased NADPH oxidase activity.
NAADP is a metabolite of NADP in vivo (Dickey et al., 1998).
It is a familiar phenomenon that exogenous and endogenous
elements play different roles in organisms. For example,
inhibition of NOS in larvae causes hypertrophy of organs and
their segments in adult flies, whereas ectopic expression of NOS
in larvae has the opposite effect (Kuzin et al., 1996). Thus, we
deduced that the high levels of endogenous NADP+ might
induce neurite degeneration and neuron apoptosis.
In summary, our results showed that, safrole oxide could
induce apoptosis of primary cultures of neurons. In the apoptotic
process, the expression of integrin β4 decreased significantly
and the levels of ROS increased dramatically. Additionally,
MnSOD activity was suppressed remarkably and the NADPH
oxidase activity was elevated dramatically in the neurons treated
with safrole oxide. The data suggested that the small molecule
safrole oxide was a powerful chemical tool for triggering
apoptosis of primary cultures of neurons. Both integrin β4 and
intracellular ROS were involved in neuronal survival and
apoptosis. In addition, the high levels of endogenous NADP+
might induce neurite degeneration. The results have led us to
pursue further research on neuronal apoptosis mediated by
integrin β4 using this powerful chemical tool.
Acknowledgements
This work was supported in part by the National Natural
Science Foundation of China (No. 30470404) and the
Foundation of the Ministry of Education (104112). We thank
Dr. Deling Yin (ETSU of USA) and Dr. Roberta Greenwood
(Shandong University of China) for critically revising the
manuscript.
Price, P., McMillan, T.J., 1990. Use of the tetrazolium assay in measuring the
response of human tumor cells to ionizing radiation. Cancer Research 50,
1392–1396.
References
Benitez, D.P., Miranda, C.L., Mendoza, B.R., Pena, C.Z., Palacios, P.E., 2003.
Prenatal and postnatal contents of amino acid neurotransmitters in mouse
parietal cortex. Developmental Neuroscience 25, 366–374.
Qato, M.K., Guenthner, T.M., 1995. 32P-postlabeling analysis of adducts
formed between DNA and safrole 2′,3′-epoxide: absence of adduct
formation in vivo. Toxicology Letters 75, 201–207.

1006
L. Su et al. / Life Sciences 80 (2007) 999–1006
Sakakura, C., Hagiwara, A., Nakanishi, M., Shimomura, K., Takagi, T.,
Yasuoka, R., Fujita, Y., Abe, T., Ichikawa, Y., Takahashi, S., Ishikawa, T.,
Nishizuka, I., Morita, T., Shimada, H., Okazaki, Y., Hayashizaki, Y.,
Yamagishi, H., 2002. Differential gene expression profiles of gastric cancer
cells established from primary tumor and malignant ascites. British Journal
of Cancer 87, 1153–1161.
Sakudo, A., Lee, D.C., Saeki, K., Nakamura, Y., Inoue, K., Matsumoto, Y.,
Itohara, S., Onodera, T., 2003. Impairment of superoxide dismutase
activation by N-terminally truncated prion protein (PrP) in PrP-deficient
neuronal cell line. Biochemical and Biophysical Research Communications
308, 660–667.
Shibata, Y., Branicky, R., Landaverde, I.O., Hekimi, S., 2003. Redox regulation
of germline and vulval development in Caenorhabditis elegans. Science
302, 1779–1782.
Smukste, I., Stockwell, B.R., 2005. Advances in chemical genetics. Annual
Review of Genomics and Human Genetics 6, 261–286.
regulation in development. International Journal of Developmental Biology
44, 725–731.
Wang, N., Du, C., Wang, S., Xie, K., Zhang, S., Miao, J.Y., 2004. D609 induces
vascular endothelial cells and marrow stromal cells differentiation into
neuron-like cells. Acta Pharmacologica Sinica 25, 442–446.
Wang, N., Lv, X., Su, L., Zhao, B.X., Zhang, S.L., Miao, J.Y., 2006. D609
blocks cell survival and induces apoptosis in neural stem cells. Bioorganic
and Medicinal Chemistry Letters 16, 4780–4783.
Weaver, V.M., Lelievre, S., Lakins, J.N., Chrenek, M.A., Jones, J.C., Giancotti,
F., Werb, Z., Bisse, M.J., 2002. Beta4 integrin-dependent formation of
polarized three-dimensional architecture confers resistance to apoptosis in
normal and malignant mammary epithelium. Cancer Cell 3, 205–216.
Whitley, M., Treloar, H., De Arcangelis, A., Labouesse, E.G., Greer, C.A., 2005.
The alpha6 integrin subunit in the developing mouse olfactory bulb. Journal
of Neurocytology 34, 81–96.
Wu, X., Lu, H., Zhou, L., Huang, Y., Chen, H., 1997. Changes of
phosphatidylcholine-specific phospholipase C in hepatocarcinogenesis and
in the proliferation and differentiation of rat liver cancer cells. Cell Biology
International 21, 375–381.
Zhang, Z., Galileo, D.S., 1998. Retroviral transfer of antisense integrin a6 or α8
sequences results in laminar redistribution or clonal cell death in developing
brain. Journal of Neuroscience 18, 6928–6938.
Zhao, B.X., Wang, D.W., Zuo, H., Qian, G.T., 2003. Synthesis and bioactivity of
1-alkoxy(phenoxy)-3-(3,4-methylenedioxyphenyl)-2-propanol. Chinese
Journal of Organic Chemistry 23, 1026–1028.
Zhao, B.X., Sha, L., Tan, W., Zuo, H., Wang, D.W., 2004a. Synthesis of
isochroman derivatives from 1-alkoxy (phenoxy)-3-(3,4-methylenedioxy)
phenyl-2-propanol by Oxa-Pictet-Spengler reaction. Chinese Journal of
Organic Chemistry 24, 1300–1303.
Zhao, Q., Araki, S., Zhang, S.L., Miao, J.Y., 2004b. Rattlesnake venom induces
apoptosis by stimulating PC-PLC and upregulating the expression of
integrin beta4, P53 in vascular endothelial cells. Toxicon 44, 161–168.
Zhao, J., Miao, J.Y., Zhao, B.X., Zhang, S.L., 2005. Upregulating of Fas,
integrin β4 and P53 and depressing of PC-PLC activity and ROS level in
VEC apoptosis by safrole oxide. FEBS Letters 579, 5809–5813.
Zuo, H., Zhao, B.X., Tan, W., Wang, D.W., Sha, L., 2004. Synthesis of 1,3-
dihydroxyacetone derivatives. China Journal of Organism Chemistry 24,
331–333.
Sommer, L., Rao, M., 2002. Neural stem cells and regulation of cell number.
Progress in Neurobiology 66, 1–18.
Spring, D.R., 2005. Chemical genetics to chemical genomics: small molecules
offer big insights. Chemical Society Reviews 34, 472–482.
Su, L., Zhao, J., Zhao, B.X., Miao, J.Y., Yin, D.L., Zhang, S.L., 2006. Safrole
oxide induced human umbilical vein vascular endothelial cell differentiation
into neuron-like cells by depressing the reactive oxygen species level at the
low concentration. Biochimica et Biophysica Acta 1763, 247–253.
Suematsu, N., Tsutsui, H., Wen, J., Kang, D., Ikeuchi, M., Ide, T., Hayashidani,
S., Shiomi, T., Kubota, T., Hamasaki, N., Takeshita, A., 2003. Oxidative
stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA
damage and dysfunction in cardiac myocytes. Circulation 107, 1418–1423.
Sunanda, B.S., Shankaranarayana, R., Raju, T.R., 1998. Corticosterone
attenuates zinc induced neurotoxicity in primary hippocampal culture.
Brain Research 791, 295–298.
Tammariello, S.P., Quinn, M.T., Estus, S., 2000. NADPH oxidase contributes
directly to oxidative stress and apoptosis in nerve growth factor-deprived
sympathetic neurons. Journal of Neuroscience 20, RC53.
Tamura, R.N., Rozzo, C., Starr, L., Chambers, J., Reichardt, L.F., Cooper, H.M.,
Quaranta, V., 1990. Epithelial integrin alpha 6 beta 4: complete primary
structure of alpha 6 and variant forms of beta 4. Journal of Cell Biology 111,
1593–1604.
Tarone, G., Hirsch, E., Brancaccio, M., De Acetis, M., Barberis, L., Balzac, F.,
Retta, S.F., Botta, C., Altruda, F., Silengo, L., 2000. Integrin function and