Toxicology 310 (2013) 92–97

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Induction of autophagy by TOCP in differentiated human

neuroblastoma cells lead to degradation of cytoskeletal components
and inhibition of neurite outgrowth
Jia-Xiang Chen a,b,c,1 , Ying-Jian Sun a,d,1 , Pan Wang a,1 , Ding-Xin Long a , Wei Li a ,
Li Li a , Yi-Jun Wu a,∗
a
Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of
Sciences, 1-5 Beichenxi Road, Beijing 100101, PR China
b
Graduate University of Chinese Academy of Sciences, Beijing 100039, PR China
c
Medical School of Nanchang University, Nanchang 330006, PR China
d
Department of Veterinary Medicine and Animal Science, Beijing University of Agriculture, Beijing 102206, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, can cause neurotoxicity such as
Received 15 April 2013 organophosphorus ester-induced delayed neuropathy (OPIDN) in humans and sensitive animals. More-
Received in revised form 22 May 2013 over, it also affects the development of central nervous system and differentiation of neuronal cells.
Accepted 24 May 2013
In this study, retinoic acid-induced differentiated human neuroblastoma SH-SY5Y cells are utilized to
Available online 3 June 2013
investigate the effects of TOCP on neurite outgrowth and the underlying mechanisms. We found that
low concentrations of TOCP induced autophagy and inhibited neurite outgrowth in a dose-dependent
Keywords:
manner with no effect on cell viability. The protein levels of high molecular weight neurofilament (NF-
Organophosphate
Neurite outgrowth
H), low molecular weight neurofilament (NF-L) and ␤-tubulin also decreased. Pretreatment cells with
Cytoskeletal component 3-methyladenine (3-MA), an autophagy inhibitor, not only inhibited the TOCP-induced autophagy, but
Degradation also reversed the inhibition of neurite outgrowth and the degradation of NF-H, NF-L, and ␤-tubulin by
Human neuroblastoma TOCP. Taken together, these results indicated that TOCP treatment induced autophagy in differentiated
SH-SY5Y cells, which lead to degradation of cytoskeletal components and inhibition of neurite outgrowth.

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction known as OP-induced delayed neuropathy (OPIDN), which affects

Tricresyl phosphate (TCP) has been widely used as plasticiz-
ers, plastic softeners, flame retardants, and jet oil additives in the
industry and tri-ortho-cresyl phosphate (TOCP) is one of the three
isomers of TCP (i.e. o-, m-, or p-cresyl) (Craig and Barth, 1999;
Winder and Balouet, 2002). Although most organophosphates
(OPs) exert their acute toxicity by suppressing acetylcholinesterase
and inducing neuromuscular block and subsequent respiratory fail-
ure, TOCP mainly induces a delayed neurodegenerative syndrome
Abbreviations: 3-MA, 3-methyladenine; CHX, cycloheximide; DMEM, Dulbecco’s
modified Eagle’s medium; ECL, enhanced chemiluminescence; NF-H, high molecular
weight neurofilament; NF-L, low molecular weight neurofilament; OP, organophos-
phates; OPIDN, organophosphorous compound-induced delayed neuropathy; PI3K,
phosphatidylinositol-3-kinase; PVDF, polyvinylidene fluoride; RA, retinoic acid;
Rapa, rapamycin; TBS, tris-buffered saline; TOCP, tri-ortho-cresyl phosphate.
∗ Corresponding author. Tel.: +86 10 64807251; fax: +86 10 64807099.
E-mail address: wuyj@ioz.ac.cn (Y.-J. Wu).
1
These authors contributed equally to this paper.
both the central and the peripheral nervous systems in humans and
sensitive species (Smith et al., 1930; Craig and Barth, 1999; Emerick
et al., 2012).
Adult hens are usually the animal models for experimental stud-
ies of delayed neurotoxicity (Abou-Donia, 1993). Another widely
used model for studying the mechanism of neurotoxicity is cell
line, such as human neuroblastoma SH-SY5Y cell line (Hong et al.,
2003; Long and Wu, 2008) and mouse neuroblastoma N2a cell line
(Fowler et al., 2001). SH-SY5Y cell is particularly useful for study-
ing neurotoxicity since it extends processes following retinoic acid
(RA) treatment and maintains many properties inherent to neuro-
blastoma cells (Hong et al., 2003; Nostrandt and Ehrich, 1992).
It has been reported that TOCP treatment can inhibit neurite
outgrowth in neuroblastoma cells (Henschler et al., 1992; Flaskos
et al., 1998; Li and Casida, 1998). Later, it was demonstrated that
loss of cytoskeletal components was the early event in the neu-
rotoxic effect of TOCP in mouse neuroblastoma N2a cells (Fowler
et al., 2001) and human neuroblastoma cells (Carlson and Ehrich,
2001; Chang and Wu, 2006), which indicated that TOCP-induced

0300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tox.2013.05.012
 J.-X. Chen et al. / Toxicology 310 (2013) 92–97
neurite outgrowth inhibition may result from the loss of cytoskele-
tal components in neuron cells. Eukaryotic cells mainly contain
three kinds of cytoskeletal components: microfilaments, interme-
diate filaments, and microtubules. Microfilaments are also named
as actin filaments, which are the thinnest filaments of the cytoskele-
ton. Microtubules are hollow cylinders about 24–26 nm in diameter
and are usually comprised of 13 protofilaments that are polymers of
alpha and beta tubulin. Intermediate filaments are structures with
8–10 nm diameter. In the nervous system, neurofilaments (NFs) are
the major intermediate filaments in neural cells. NFs are formed by
the neurofilament triplet proteins: low molecular weight neuro-
filament subunit (NF-L), middle molecular weight neurofilament
subunit (NF-M), and high molecular weight neurofilament sub-
unit (NF-H) (Lee and Cleveland, 1994; Liem and Messing, 2009).
However, little is known about the underlying mechanism of TOCP-
induced loss of cytoskeletal components.
Macroautophagy, hereafter referred to as autophagy, is an evo-
lutionarily conserved mechanism in all eukaryotes. Its basic role
is the bulk turnover of proteins and intracellular organelles in
response to nutrient starvation (Klionsky and Emr, 2000; Tsujimoto
and Shimizu, 2005). It involves the formation of double-membrane
structures, termed autophagosomes or autophagic vacuoles, which
fuse with the lysosomes to form the autolysosomes to degrade
their contents (Klionsky and Emr, 2000). Autophagy plays an essen-
tial role in differentiation and development, as well as in cellular
response to stress. It is activated during amino-acid deprivation
and has been associated with neurodegenerative diseases, cancer,
pathogen infections and myopathies (Cuervo, 2004; Shintani and
Klionsky, 2004).
The aim of the present study is to investigate whether degra-
dation of cytoskeletal components by TOCP is associated with
induction of autophagy, which subsequently leads to inhibition of
neurite outgrowth in differentiated SH-SY5Y cells.
2. Materials and methods
2.1. Materials
The human neuroblastoma SH-SY5Y cell line was purchased from the Cell Center
of Chinese Academy of Medical Sciences (Beijing, China). TOCP (purity >99%) was
purchased from BDH Chemical Ltd. (Poole England, UK). The transfection reagent
Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Groningen,
the Netherlands). GFP-LC3 plasmid was constructed in our laboratory. Retinoic acid
(RA), cycloheximide (CHX), 3-methyladenine (3-MA), rapamycin (Rapa) and mouse
anti-NF-H monoclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Rabbit anti-LC3 monoclonal antibody, mouse anti-Atg5 monoclonal antibody,
mouse anti-Beclin 1 monoclonal antibody, mouse anti-NF-L monoclonal antibody,
rabbit anti-␤-tubulin monoclonal antibody, mouse anti-GAPDH monoclonal anti-
body, rabbit anti-␤-actin monoclonal antibody, goat anti-mouse IgG-HRP and goat
anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Pierce
Biotechnology (Rockford, IL, USA).
2.2. Cell culture and transfection
Cells were grown and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and
100 ␮g/ml streptomycin. Incubations were carried out at 37 ◦ C in a humidified atmo-
sphere of 5% CO2 /95% air. The cells were maintained in the logarithmic phase of
growth and sub-cultured at 3–4 days intervals. Transient transfection of GFP-LC3 in
SH-SY5Y cells was performed using Lipofectamine 2000 according to the manufac-
turer’s protocol.
2.3. Cell differentiation
Cells were induced to differentiate with DMEM medium containing 20 ␮M RA in
the dark for 4 days. Differentiated cells were treated with 0–1.0 mM TOCP for 24 h,
then cells were washed in Tris-buffered saline (TBS; 50 mM Tris–HCl and 150 mM
NaCl, pH 7.4) before fixation with ice-cold 90% (v/v) methanol in TBS at −20 ◦ C for
20 min. Cells were stained using Coomassie brilliant blue for 10 min at room temper-
ature, and then viewed using an inverted phase-contrast microscope (DMRBE, Leica).
Cells were considered to be differentiated if they had at least one process longer
than the cell body, which could be regarded as a neurite (Raghunath et al., 2000).
93
The length of the longest neurite was measured in at least 100 cells in randomly
chosen fields with an inverted microscope. At least three independent experiments
were conducted and the results are expressed as mean ± standard error (S.E.).
2.4. Cell treatment
Differentiated cells were pretreated with 30 ␮M cycloheximide (CHX) and then
treated with 0–1.0 mM TOCP for 24 h in the absence or presence of 1.0 mM 3-MA.
3-MA was freshly dissolved in culture medium 30 min before use. Other reagents
were dissolved in DMSO. Cells were treated with 10 nM rapamycin (Rapa) or starved
as positive controls for autophagy. For starvation experiments, cells were cultured
in Hanks’ solution for 6 h in the presence of 30 ␮M CHX.
2.5. Western blotting analysis
Cells were trypsinized, washed twice with ice-cold PBS, and harvested in RIPA
buffer (50 mM Tris pH 7.5, 0.3 M NaCl, 5 mM ethyleneglycotetraacetic acid, 1 mM
ethylenediaminetetraacetic acid, 0.5% Triton X-100, 0.5% NP-40, 0.1 mM phenyl-
methylsulphonyl fluoride, and 10 ␮g/ml each of aprotinin, leupeptin and pepstatin),
and then sonicated on ice. Lysates were clarified, and the protein concentrations
were determined according to the method of Lowry et al. (1951) using bovine serum
albumin (BSA) as a standard. The protein samples were separated by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis with a 5% stacking gel and 8–15% sep-
arating gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore
Corporate, Billerica, MA, USA). Following transfer, membranes were blocked with
Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat milk for at least
1 h at room temperature, and incubated with primary antibodies (diluted 1:1000),
and then with appropriate secondary antibodies conjugated to horseradish perox-
idase (diluted 1:5000). Immunoreactive bands were detected with ChemiDocXRS
(Bio-Rad, Hercules, CA, USA) using standard ECL reagents (Pierce Biotechnology,
Rockford, IL, USA). The blots were digitalized, and the protein levels were quantified
by directly relating antibody reactivity to level of specific protein using the Quantity
One software program.
2.6. Statistical analysis
Values were expressed as the means ± SEM. Data were evaluated by one-way
analysis of variance (ANOVA) with Newman–Keuls multiple range test using SPSS
12.0 statistical software. For each test, P-values less than 0.05 (P < 0.05) were con-
sidered statistically significant.
3. Results
3.1. TOCP induces autophagy in differentiated SH-SY5Y cells
To evaluate whether TOCP induces autophagy in differentiated
SH-SY5Y cells, the cells were induced to differentiate with 20 ␮M
RA for 4 days, then transfected with GFP-LC3 and treated with dif-
ferent concentrations of TOCP (0, 0.5, 1.0 mM) and 3-MA (1.0 mM)
plus TOCP (1.0 mM) for 24 h. Autophagic vesicles were visualized by
confocal microscope. We found that autophagic vesicles increased
significantly after cells were treated with TOCP, indicating that
TOCP can induce autophagy in differented SH-SY5Y cells (Fig. 1).
We also analyzed the autophagy protein LC3, a widely used
marker of mammalian autophagy (Kabeya et al., 2000). During the
induction of autophagy, cytosolic form of LC3 (LC3-I) undergoes
covalent conjugation with phosphatidylethanolamine to form a
faster SDS-PAGE migrating form (LC3-II), which is recruited and
bound to autophagosome membrane (Kabeya et al., 2000). The con-
version of LC3-I into LC3-II was considered as a general marker for
the initiation of autophagy (Mizushima, 2004). The amount of the
membrane-bound form of LC3-II is correlated with the extent of
autophagosome formation (Kabeya et al., 2000). As shown in Fig. 2,
after treatment with TOCP, both LC3-II and the ratio of LC3-II/LC3-
I were markedly increased. Beclin-1 is a mammalian ortholog of
the yeast autophagy-related gene (Atg) 6 and it is required for the
initiation of the autophagosome formation. Atg5 is necessary for
autophagy due to its important role in autophagosome elonga-
tion. Autophagy proteins Atg5 and Beclin 1 were also increased
after treated with TOCP, indicating TOCP could induce autophagy
in the cells. After cells were pretreated with 3-MA, an inhibitor
of autophagy by blocking autophagosome formation via the inhi-
bition of type III phosphatidylinositol-3-kinase (PI3K) (Seglen and
94 J.-X. Chen et al. / Toxicology 310 (2013) 92–97

Fig. 1. TOCP induces autophagy in differentiated SH-SY5Y cells. Differentiated cells were transfected with GFP-LC3 and treated with different concentrations of TOCP (0,
0.5, 1.0 mM) and 3-MA (1.0 mM) plus TOCP (1.0 mM) for 24 h. Starvation and rapamycin treatment were used as positive controls for autophagy. Cells were visualized under
a TCS-4D laser scanning confocal microscope (1000×). The treatment groups are: (A) 0 mM TOCP; (B) starvation; (C) rapamycin; (D) 0.5 mM TOCP; (E) 1.0 mM TOCP; (F)
1.0 mM 3-MA plus 1.0 mM TOCP. The experiments were done in triplicate and repeated three times. Data were analyzed by one-way ANOVA. The results are presented as
mean ± SEM with n = 3. ‘*’ indicates significant difference from control group with P < 0.05.

Gordon, 1982), both LC3-II and the ratio of LC3-II/LC3-I were dra-
matically decreased in the cells treated with TOCP. And Atg5 and
Beclin 1 were also decreased, implicating that 3-MA could inhibit
the autophagy induced by TOCP.
To determine whether 0.2–1.0 mM TOCP can lead to cell death,
cell viability was determined by MTT assay. We found that treat-
ment with 0.2–1.0 mM TOCP for 24 h did not reduce cell viability
(data not shown).

3.2. TOCP inhibits neurite outgrowth in differentiated SH-SY5Y

cells via induction of autophagy

To study the role of TOCP in neurite outgrowth, differentiated

Fig. 2. 3-MA inhibits autophagy induced by TOCP in differentiated SH-SY5Y cells.
Differentiated cells were treated with 0–1.0 mM TOCP for 24 h in the absence or pres-
ence of 1.0 mM 3-MA. The protein levels of Atg 5, Beclin 1 and LC3 were determined
by Western blotting analysis. GAPDH was used as a loading control. The experiments
were done in triplicate and repeated three times.
SH-SY5Y cells were treated with 0–1.0 mM TOCP for 24 h. As shown
in Fig. 3A and C, the differentiated cells displayed profound mor-
phological alterations characterized by the appearance of axon-like
processes, a typical neuronal phenotype. And TOCP inhibited neu-
rite outgrowth of the cells, indicated by the average length of
axon-like processes, in a dose-dependent manner. The average
lengths of neurites of differentiated cells treated with TOCP at the
concentrations of 0.2, 0.5, and 1.0 mM were 92.6 ± 6.5, 65.2 ± 7.4,
and 43.2 ± 8.6 ␮m, respectively. In contrast, the average length of
 J.-X. Chen et al. / Toxicology 310 (2013) 92–97 95

Fig. 3. 3-MA reverses the inhibition of neurite outgrowth by TOCP treatment in differentiated SH-SY5Y cells. Differentiated cells were treated with 0–1.0 mM TOCP for 24 h
in the absence (A and C) or presence (B and D) of 3-MA and then were fixed in methanol and stained with Coomassie brilliant blue. Axon lengths were quantified using an
inverted light microscope. Data were analyzed by one-way ANOVA. The results are presented as mean ± SEM with n = 3. ‘*’ indicates significant difference from control group
with P < 0.05.

neurites in untreated differentiated cells was 105.3 ± 5.1 ␮m. Fur-
thermore, as shown in Fig. 3B and D, in the presence of 3-MA,
the average length of neurites in differentiated cells treated with
TOCP had no significant difference with control untreated cells. This
result suggests that the inhibition of neurite outgrowth by TOCP
was mediated by induction of autophagy.
3.3. TOCP induces the degradation of cytoskeletal components in
differentiated SH-SY5Y cells via autophagy
To study the effect of TOCP on cytoskeletal components, differ-
entiated SH-SY5Y cells were treated with 0–1.0 mM TOCP for 24 h in
the presence of 30 ␮M CHX. As shown in Fig. 4, TOCP treatment sig-
nificantly decreased the protein levels of NF-H, NF-L, and ␤-tubulin
with no effect on the protein level of ␤-actin in differentiated SH-
SY5Y cells. On the contrary, in the presence of 3-MA, there were no
significant changes observed on the protein levels of NF-H, NF-L,
␤-tubulin and ␤-actin in the CHX-pretreated cells after incubation
with 0–1.0 mM TOCP for 24 h. These data suggest that TOCP might
induce the degradation of the cytoskeletal proteins such as NF-H,
NF-L and ␤-tubulin, and the degradation of cytoskeletal proteins in
differentiated SH-SY5Y cells was mediated by autophagy.
4. Discussion
In the present study, we showed that TOCP induced autophagy
in differentiated SH-SY5Y cells, which caused subsequent degra-
dation of cytoskeletal components and inhibition of neurite
outgrowth.
TOCP is widely used in the industry and its toxicity has drawn
serious concerns (Craig and Barth, 1999; Winder and Balouet,
2002; Inoue et al., 1988). It has been used as the prototype
neuropathy-inducing agent for experimental studies of OPIDN.
TOCP at low level inhibited neurite outgrowth in differentiated
N2a and PC12 cells (Fowler et al., 2001; Flaskos et al., 1998; Li
and Casida, 1998). SH-SY5Y cells can extend and maintain axon-
like processes after induction with RA, and are useful in vitro cell
models for studying mechanisms of neurotoxicity.
Autophagy is a major catabolic pathway in all eukaryotes for
degradation of proteins and organelles in response to nutrient star-
vation (Klionsky and Emr, 2000; Tsujimoto and Shimizu, 2005).
Accumulating evidence shows that autophagy has been associated
with neurodegenerative diseases. For example, the induction of
autophagy in axonal dystrophy and degeneration in Purkinje cells
were observed during excitotoxic neurodegeneration (Wang et al.,
2006). It was reported that induction of autophagy caused neurite
degeneration in mouse superior cervical ganglion neurons (Yang
et al., 2007). In addition, the accumulation of autophagosomes has
been found in neurites in a transgenic mouse model of Alzheimer’s
disease (Yu et al., 2005) and in substantia nigra neurons from
patients with Parkinson’s disease (Zhu et al., 2003). These above
studies indicate a close relationship between the autophagic pro-
cess and axonal degeneration. However, emerging evidence shows
that the loss of autophagy in the mouse central nervous system
caused neurodegeneration, indicating that basal autophagic activ-
ity is essential for the survival of neuronal cells (Hara et al., 2006;
Komatsu et al., 2006).
To evaluate whether TOCP can induce autophagy in differen-
tiated SH-SY5Y cells, we transiently transfected GFP-LC3 into the
differentiated SH-SY5Y cells and we found that autophagic vesi-
cles increased significantly after treatment with TOCP. TOCP also
markedly increased LC3-II, the ratio of LC3-II/LC3-I and autophagy
protein atg5 and Beclin 1, indicating that TOCP could induce
autophagy in the cells. mTOR is a key regulator of autophagy, whose
96 J.-X. Chen et al. / Toxicology 310 (2013) 92–97

Fig. 4. 3-MA inhibits the degradation of cytoskeletal proteins by TOCP treatment in differentiated SH-SY5Y cells. Differentiated cells were treated with 0–1.0 mM TOCP for
24 h in the absence (A and B) or presence (C and D) of 3-MA. The protein levels of NF-H, NF-L, ␤-tubulin and ␤-actin were determined by immunoblotting with specific
antibodies. GAPDH was used as a loading control. Data were analyzed by one-way ANOVA. The results are presented as mean ± SEM with n = 3. ‘*’ indicates significant
difference from control group with P < 0.05.

activation is controlled by phosphoinositide 3-kinase (PI3K). 3-MA
is a specific inhibitor of PI3K activity, which is one of the most
widely used inhibitors of the initial phase of the autophagic pro-
cess: the sequestering of cytoplasmic material by the lysosome
(Seglen and Gordon, 1982). 3-MA, an autophagy inhibitor, could
dramatically decrease the autophagic vesicles, the LC3-II level, the
ratio of LC3-II/LC3-I and the protein levels of Atg5 and Beclin 1,
implicating 3-MA could inhibit the autophagy induced by TOCP.
We found that 0.2–1.0 mM TOCP had no effect on the cell viabil-
ity. Indeed, our earlier studies showed that treatment with TOCP at
higher concentrations or for longer time could significantly inhibit
cell viability of SH-SY5Y cells (Chang and Wu, 2006; Long and Wu,
2008).
We also showed that neurite outgrowth was inhibited by TOCP
in a dose-dependent manner. 3-MA reversed the inhibition of neu-
rite outgrowth by TOCP in the cells, indicating that autophagy
induced by TOCP played an important role in the inhibition of neu-
rite outgrowth.
Williamson et al. (1996) has showed that cytoskeletal compo-
nents play an important role in the control of axon growth and
stability, and alteration of cytoskeletal components is associated
with neurodegenerative diseases. Studies have demonstrated that
inhibition of neurite outgrowth by TOCP is associated with disrup-
tion of cytoskeletal network (Fowler et al., 2001; Flaskos et al., 1998;
Li and Casida, 1998) and loss of cytoskeletal components (Flaskos
et al., 1998; Carlson and Ehrich, 2001). In this study, we found that
treatment with TOCP significantly decreased the protein levels of
NF-H, NF-L and ␤-tubulin with no effect on the protein level of ␤-
actin. The results indicated that TOCP-induced inhibition of neurite
outgrowth in differentiated SH-SY5Y cells might result from loss
of cytoskeletal proteins such as NF-H, NF-L and ␤-tubulin. Mean-
while, 3-MA, an inhibitor of autophagy, reversed degradation of the
cytoskeletal proteins such as NF-H, NF-L, and ␤-tubulin by TOCP
treatment.
Taken together, we found that inhibition of neurite outgrowth
by TOCP may result from the degradation of cytoskeletal proteins
such as NF-H, NF-L, and ␤-tubulin in differentiated human neuro-
blastoma SH-SY5Y cells by induction of autophagy.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported in part by the grants from National
Natural Science Foundation of China (no. 31071919) and the
National Basic Research Program of China (no. 2012CB114100).
 J.-X. Chen et al. / Toxicology 310 (2013) 92–97
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