Toxicology 262 (2009) 258–264

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

Phenylmethylsulfonyl fluoride protects against the degradation of

neurofilaments in tri-ortho-cresyl phosphate (TOCP) induced
delayed neuropathy
Fuyong Song a,1 , Yongjian Yan b,1 , Xiulan Zhao a , Dandan Dou a , Cuili Zhang a , Keqin Xie a,∗
a
Institute of Toxicology, Shandong University, 44 West Wenhua Road, Jinan, Shandong 250012, PR China
b
Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences, 89 Jingshi Road, Jinan, Shandong 250012, 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) is an organophosphorus ester, which can cause a type of neurotox-
Received 5 May 2009 icity known as organophosphate-induced delayed neuropathy (OPIDN). Our recent study has shown
Received in revised form 18 June 2009 that the enhanced degradation of neurofilament (NF) in peripheral nerve of hens is an early event of
Accepted 19 June 2009
TOCP-induced OPIDN (Song et al., 2009). The main objective of this investigation is to study the effect of
Available online 30 June 2009
TOCP administration on NF content and NF degradation when OPIDN is blocked by pretreatment with
phenylmethylsulfonyl fluoride (PMSF). The hens were pretreated 24 h earlier with PMSF and subsequently
Keywords:
treated with a single dosage of 750 mg/kg TOCP, then sacrificed on the corresponding time points of 0,
Tri-ortho-cresyl phosphate (TOCP)
Neurofilament
1, 5, 10, and 21 days after dosing TOCP, respectively. The tibial nerves were dissected, homogenized, and
Organophosphorus ester-induced delayed centrifuged at 100,000 × g. The level of NF triplet protein in both pellet and supernatant fractions of tibial
neuropathy (OPIDN) nerves was determined. Western blotting analysis showed a significant increase of three NF subunits in
Phenylmethylsulfonyl fluoride hens treated with PMSF and TOCP compared with the control. These changes were observed within 24 h
of PMSF administration and then followed by an obvious recovery. Furthermore, accompanied with the
increase of NF content, a significant decline in NF-L degradation rate was observed in both fractions of
tibial nerves. Taken together, these results demonstrated the pretreatment with PMSF could inhibit TOCP-
induced NF degradation while it protected hens against the development of OPIDN, which suggested the
inhibition of NF-associated protease in peripheral nerves might be an underlying protective mechanism
of PMSF against OPIDN.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction OPIDN can be classified as a distal sensorimotor axonopathy.

Organophosphorus compounds (OP) are widely used in agricul-
ture and industry and some of these chemicals can cause central and
peripheral distal axonopathy known as organophosphate-induced
delayed neuropathy (OPIDN) in humans and other sensitive species
(Abou-Donia, 1981, 2003). Typically, the delayed neuropathy occurs
2–3 weeks after a single exposure of organophosphate compound
when signs of both the acute cholinergic and the intermediate
syndromes have subsided. Signs and symptoms of OPIDN include
tingling of the hands and feet, followed by sensory loss, progres-
sive muscle weakness and flaccidity of the distal skeletal muscles
of the lower and upper extremities, and ataxia (Lotti, 1992; Lotti
and Moretto, 2005).
∗ Corresponding author. Tel.: +86 531 88382132; fax: +86 531 88382553.
E-mail address: keqinx@sdu.edu.cn (K. Xie).
1
These authors contributed equally to this work.
The morphological pattern of OPIDN consists of symmetrical, distal
axonal degeneration of ascending and descending nerve fiber tract
located in central and peripheral nervous systems (Abou-Donia and
Graham, 1979; Tanaka and Bursian, 1989; Classen et al., 1996). At the
ultrastructural level, the axonal swelling containing aggregations
of neurofilaments (NFs), microtubules, multivesicular vesicles, and
proliferation of smooth endoplasmic reticulum are presented in
early stage, and followed by partial matting, and disappearance of
NFs from swollen axons (Bischoff, 1970).
Although the mechanism of OPIDN has been investigated for
some decades, little is known regarding early events in OPIDN
except for the essential inhibition of neurotoxic esterase (NTE)
(Johnson, 1982). Generally, the inhibition by approximately 75%
or greater of whole hen brain NTE 1 day after dosing is used as
an indicator of neurotoxic potential. However, the physiological
functions and biochemical basis for neurodegeneration of NTE are
unknown. Hence, the most crucial issues in the mechanisms of
OPIDN development and progression remain obscure. Consider-
ing the pathological characteristics with NFs changes in OPIDN,

0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2009.06.018
 F. Song et al. / Toxicology 262 (2009) 258–264
we hypothesized that a disturbance of NFs homeostasis produced
by OP might participate in the progression of OPIDN. NFs, belong-
ing to the type IV intermediate filaments, are classed into three
distinct groups according to their molecular masses: NF-H (heavy
chain), NF-M (middle chain) and NF-L (light chain) (Lee et al.,
1993). Together with microtubules and other associated proteins,
NFs make up the dynamic axonal cytoskeleton. NFs are essential
elements for establishing the correct diameters of large myeli-
nated motor and sensory axons, and the establishment of axon
caliber is important for normal function of nervous system (Lee
and Cleveland, 1994).
The previous researches in this laboratory demonstrated that
the exposure to tri-ortho-cresyl phosphate (TOCP) resulted in sig-
nificant changes of NF contents in the central and peripheral nerve
tissues of hens (Zhao et al., 2004, 2006). Furthermore, our recent
study has showed a marked enhancement of NF-L degradation in
tibial nerves as early as 24 h after TOCP dosing. The disruption of
NF homeostasis in peripheral nerves induced by TOCP could be
ascribed to the enhancement of NFs degradation (Song et al., 2009).
These findings suggested that NF degradation was an early molec-
ular event in TOCP-induced delayed neuropathy, which might be
implicated in the development of OPIDN.
Certain esterase inhibitors such as phenylmethanesulfonyl flu-
oride (PMSF) and phenyl N-methyl N-benzyl carbamate (PMBC)
were found to be protective when given prior to organophospho-
rus esters. Pretreatment of hens with PMSF prior to TOCP has been
shown to be effective in preventing the signs and symptoms of
the delayed TOCP neuropathy (Carrington and Abou-Donia, 1983).
Furthermore, it has also been demonstrated that pretreatment of
hens with PMSF is effective in preserving motor nerve terminal
function, electrophysiologic and ultrastructural neuropathological
manifestations against the effects of delayed neuropathy caused by
OP (Baker et al., 1980; Drakontides and Baker, 1983; Massicotte et
al., 1999). However, whether PMSF also prevent NF degradation in
nerve tissues induced by TOCP is unknown.
Therefore, in the present investigation, we have investigated the
time course of TOCP-induced NFs alteration in tibial nerves of hens
pretreated with PMSF, and studied the alteration of NF degradation
during the prevention of OPIDN by PMSF.
2. Materials and methods
2.1. Materials
Tri-ortho-cresyl phosphate (TOCP, purity > 99%), phenylmethanesulfonyl fluo-
ride (PMSF), and Protease Inhibitor Cocktail set III were purchased from Merck
Biosciences, Inc. (Darmstadt, GER). Monoclonal antibodies anti-NF-H (clone NE-14),
anti-NF-M (cloneNN-18), anti-NF-L (clone NR-4), anti-␤-actin (clone AC-15), and
HRP-conjugated goat-anti-mouse IgG were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). BCATM Protein assay Kit and SuperSignal® West Pico Chemilumi-
nescent Substrate Kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL,
USA). Film was obtained from Eastman Kodak Co. (Rochester, NY, USA). All other
chemicals were of highest quality commercially available.
2.2. Animal treatment and neurological testing
Roman hens, 10 months old and weighing 1.5–2.0 kg, obtained from Institute of
Poultry, Academy of Agriculture of Shandong (Jinan, CN), were used in this study.
Drinking water and complete-value hen powder food were available ad libitum. The
hens were housed one per cage made of stainless steel wire. The animal room was
maintained at approximately 22 ◦ C and 50% humidity with a 12-h light/dark cycle.
All experiments were carried out in accordance with the NIH Guide for Care and
Use of Laboratory Animals and followed the principles in the “Use of Animals in
Toxicology”. After 7 days for acclimatiation, the animals were randomly divided into
six groups, i.e. five experimental groups (0-day, 1-day, 5-day, 10-day, and 21-day
groups; n = 6 each group) and one control group (n = 6).
In order to evaluate the time-dependent effect of PMSF on the changes of NFs
induced by TOCP, hens in experimental groups were treated with PMSF and TOCP.
PMSF was dissolved in DMSO and injected subcutaneously (s.c.) in the anterotho-
racic region of hens with a dose of 60 mg/kg in 0.2 ml/kg. After 24 h of PMSF dosing,
hens in experimental groups were also administered by gavage with a single dose
of 750 mg/kg TOCP. TOCP was dissolved in corn oil and administered at 0.65 ml/kg.
259
The control group hens received an equivalent volume of corn oil. Birds in different
groups were anesthetized with ether and sacrificed by decapitation on the corre-
sponding time points of 0, 1, 5, 10, and 21 day of TOCP exposure, respectively. The
tibial nerves were dissected from the ankle to the trifurcating point where it divides
into two large branches (the tibial and common peroneal nerves), frozen in liquid
nitrogen, and then kept at −80 ◦ C until used for the determination of NFs proteins.
During the course of experiment, the hens were weighed twice a week and
examined daily for the clinical signs of delayed neurotoxicity after the administration
of TOCP. Neurological evaluation was performed by a blinded observer who was not
involved in animal care and administration. OPIDN neurological signs were assessed
by an eight-point graded scale (0, normal ambulation; 1–2, slight and infrequent
hindlimb incoordination; 3–4, moderate but definite hindlimb incoordination; 5–6,
severe and frequent difficulty in walking and standing erect; 7–8, virtual to complete
hindlimb paralysis) (Pope and Padilla, 1990).
2.3. Tissue preparation, electrophoresis and immunoblotting
Tibial nerves were broken into powder with pestle in liquid nitrogen, and
homogenized in ice-cold buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5),
25 mM KCl, 2 mM MgCl2 , 5 mM EGTA, 5 mM dithiothreitol, Protease Inhibitor Cock-
tail (50 ␮l/g tissue) and phosphatase inhibitors (5 mM Na3 VO4 , 10 mM Na4 P2 O7 and
1 mM iodoacetic acid). Nerve homogenates were centrifuged at 100,000 × g for 1 hr
to yield a high-speed pellet (P) and a high-speed supernatant (S) fraction (Tsuda et al.,
1997; Lopachin et al., 2005). Protein concentration of two fractions was determined
by using BCATM Protein assay Kit.
To assess relative changes of protein content in tissue preparations, correspond-
ing protein samples from both control and experimental animals were subjected
to SDS-PAGE on 4% stacking and 7.5% or 10% resolving gel. Following electrophore-
sis, proteins were transferred electrophoretically to nitrocellulose membranes. Then
the membranes were blocked with 4% fat-free milk for 45 min and incubated with
primary antibody diluted in 0.1% BSA for 3 h. Following primary antibody, mem-
branes were washed in TBS and incubated with horseradish peroxidase-conjugated
secondary antibody for 3 h at the room temperature. After being washed again, the
membranes were incubated by using the SuperSignal West Pico Chemiluminescent
Substrate reagents for 5 min, and then exposed to film for 15 s. Immunoreactive
bands of proteins were scanned with Agfa Duoscan T1200 scanner and digitized data
were quantified as integrated optical density (IOD) using Kodak Imaging Program
and Image-Pro Plus software.
2.4. Statistical analysis
The data of IOD value of immunoreactive bands were expressed as mean ± S.D.,
statistical analysis was performed with one-way analysis of variance (ANOVA), fol-
lowed by LSD’s post hoc tests, which was provided by SPSS 10.0 statistical software.
The differences were significant at P < 0.05.
3. Results
3.1. Clinical signs
A single dose of 750 mg/kg TOCP can induce a delayed neu-
ropathy in hens. The results have been reported in our previous
study (Song et al., 2009). The TOCP-treated hens exhibited slightly
abnormal gait firstly on day 5, and then the symptoms aggra-
vated progressively. Most of the hens reached hindlimb paralysis
on day 15 post-dosing (mean clinical score = 7.5). By the end of
21-day experimental period, all birds showed complete paraly-
sis gait (mean clinical score = 8.00). In contrast, pretreatment of
hens with PMSF (60 mg/kg, s.c.) 24 h before the administration of
TOCP protected the hens against the delayed neuropathy. No signif-
icant clinical neurotoxic signs were observed through experimental
period. Furthermore, no severe signs of acute intoxication were evi-
dent in all hens of experimental groups. On day 21 post-dosing, the
mean body weight of experimental groups did not show significant
changes compared with that of control hens.
3.2. Alterations of NF subunits in the supernatant and pellet
fractions of tibial nerves
In order to evaluate the effect of PMSF on the NFs in peripheral
nerves of hens treated with TOCP, we analyzed the protein levels of
three NF subunits in supernatant and pellet fractions of hen tibial
nerves by western blotting.
260 F. Song et al. / Toxicology 262 (2009) 258–264

Fig. 1. Time-course alteration of NF subunit contents in the supernatant fraction of tibial nerves of hens pretreated with PMSF 24 h earlier and subsequently TOCP. Represen-
tative immunoblots of NFs are also shown below the graph. The control is presented with 100%, the level of the test groups is described with the percentage of the control.
The results are presented as a mean percentage of the corresponding control ± S.D. Statistical significance is determined by using one-way analysis of variance (ANOVA). The
asterisk indicates statistical difference (**P < 0.01).

As shown in Fig. 1, NF-H content in supernatant fraction of tibial
nerves of treated animals demonstrated a trend of significant ele-
vation. When compared with the control, NF-H content increased
by 76.5% and 36.2% (P < 0.01) on day 0 and 1 post-dosing TOCP,
respectively. Subsequently, NF-H in tibial nerve showed a gradual
recovery as the time went on. On day 5, 10, and 21 post-dosing
TOCP, the level of NF-H return to that of the control. When focused
on NF-M, it was shown that NF-M was markedly elevated in the
supernatant of tibial nerves as early as day 1 following PMSF admin-
istration. The content of NF-M increased by 187.0% (P < 0.01) on
day 0 of the experiment. Subsequently, it began to decrease, but
still remained the level of 145.8%, 113.4%, 71.5%, and 49.6% more
than that of the control on day 1, 5, 10, and 21 post-dosing TOCP,
respectively (P < 0.01). Furthermore, an analogous change in NF-L
content was also observed. In comparison with the control, the level
of NF-L in the supernatant increased by 117.3%, 90.5%, 84%, 85.2%
and 41.8% (P < 0.01) on day 0, 1, 5, 10, and 21 of the experiment,
respectively.
To determine whether alteration of NF subunits was due to any
redistribution among subcellular fractions induced by TOCP, the
content of NFs in the pellet fraction of tibial nerves was also exam-
ined. As shown in Fig. 2, NF-H content in pellet fraction of treated
animals exhibited a similar change to that in supernatant fraction.
NF-H increased by 36.7% and 24.2% (P < 0.01), respectively, on day 0
and 1 post-dosing TOCP. Furthermore, the protein level of NF-M in
the pellet fraction increased by 61.7% on day 1 post-dosing PMSF.
This dramatic increase was followed by an obvious recovery. The
level of NF-M in the pellet fraction retuned to 130.4%, 93.4%, 109.5%
and 96.6% of the control on day 1, 5, 10, and 21 post-dosing TOCP,
respectively (P < 0.01). In addition, a similar altered pattern of NF-L
was also observed. The level of NF-L in the pellet fraction increased
by 16.7% and 20.7% (P < 0.01) on day 0 and 1 of the experiment,
respectively.
3.3. Alterations of NF-L degradation in the supernatant and pellet
fractions of tibial nerves
Since there was a significant increase in levels of three NF sub-
units in the supernatant and pellet fractions of tibial nerves, we
attempted to determine whether the alteration of NFs was asso-
ciated with the inhibition of NF degradation. In this study, we
detected the effect of PMSF pretreatment on NF-L degradation in
tibial nerves of hens exposed to TOCP.
As we expected, a decline in NF-L degradation was observed in
both fraction of tibial nerves of hens pretreated with PMSF and sub-
sequently with TOCP. Western blotting analysis showed the rate of
NF-L degradation in the supernatant of tibial nerves decreased from
47.7% in the controls to 26.8%, 28.6%, 35.9% and 40.4% (P < 0.01) on
day 0, 1, 5, and 10 post-dosing TOCP, respectively, and then returned
back to 46.6% on day 21 (Fig. 3). Furthermore, the rate of NF-L degra-
dation in the pellet of tibial nerves decreased from 37.8% in the
controls to 13.3%, 13.2%, 15.6%, and 31.2% (P < 0.01) on day 0, 1, 5,
and 10 post-dosing TOCP, respectively, and recovered to 36.2% on
day 21 post-dosing TOCP.
In short, the pretreatment of PMSF resulted in a significant
reduction of NF-L degradation rate in tibial nerve of hens, among
which the changes of NF degradation in the supernatant of tibial
nerves appeared to be more noticeable than that in the pellet, and
changes in the early stage after PMSF administration appeared to
be more noticeable than that in the late stage.
4. Discussion
Organophosphate-induced delayed neuropathy (OPIDN) is an
axonal degeneration of peripheral nerves and spinal cord caused by
certain organophosphorus compounds. In a recent study, we have
investigated the time course of alterations of NFs proteins in tib-
 F. Song et al. / Toxicology 262 (2009) 258–264 261

Fig. 2. Time-course alteration of NF subunit contents in the pellet fraction of tibial nerves of hens pretreated with PMSF 24 h earlier and subsequently TOCP. Representative
immunoblots of NFs are also shown below the graph. The control is presented with 100%, the level of the test groups is described with the percentage of the control. The
results are presented as a mean percentage of the corresponding control ± S.D. Statistical significance is determined by using one-way analysis of variance (ANOVA). The
asterisk indicates statistical difference (**P < 0.01).

ial nerves during the process of TOCP-induced OPIDN (Song et al.,
2009). The results demonstrated that TOCP intoxication resulted in
significant alterations in NF subunits contents in both fractions of
tibial nerves, among which a nearly depletion of NF-M and NF-L was
observed as early as 24 h after TOCP dosing. Furthermore, quanti-
tative results revealed the rate of NF-L degradation was inversely
correlated to the level of NF-L in tibial nerves. As a result, we specu-
lated that increased NF degradation was an early molecular event in
the onset and development of TOCP-induced delayed neurotoxicity.
According to the hypothesis of NTE, NTE is thought to be the
molecular target and neuropathy to be initiated with a two-step
mechanism: progressive inhibition of NTE and aging of the phos-
phorylated enzyme (Johnson, 1982). Other chemicals such as PMSF
producing an inhibited NTE, which is incapable of aging, were
thought to be non-neuropathic. When given before a challenging
dose of a neuropathic organophosphate, PMSF is able to protect
animas from neuropathy. The protection has been speculated to be
the result of occupancy of the NTE active site by PMSF, thus prevent-
ing covalent binding of the neurotoxic OP ester (Lotti and Moretto,
2005). However, NTE and the proposed mechanism of OPIDN have
been challenged since this viewpoint was proposed. Several lines
of evidence supported that aging may not always be essential in
causing neuropathy. In fact, mipafox and methamidophos form an
inhibited NTE which apparently does not age and yet produces
neuropathy (Lotti et al., 1993).
Considering that increased NF degradation might be involved in
the onset and development of TOCP-induced OPIDN and PMSF can
protect against TOCP-induced OPIDN, we speculated that the pro-
tection of PMSF might be associated with its activity of inhibiting
NF degradation in OPIDN. In the present investigation, hens that
were pretreated 24 h earlier with PMSF and subsequently received
TOCP did not develop any clinical signs of the delayed neuropathy
as expected. In the meantime, western blotting analysis showed
an obvious increase of three NF subunits in both fractions of tibial
nerves of hens. These changes were observed within 24 h of PMSF
administration, and appeared to be more noticeable in the early
stage of the experiment than in the late stage. As for underlying
reason for this phenomenon, it might be related to the diminishing
effect of PMSF as the time went on. Since the drug is very unsta-
ble in aqueous solution, it was inevitably broken down in vivo.
Consequently, the substantial effect of PMSF on NF would sub-
side. Furthermore, accompanied with the increase of NF content,
the rate of NF-L degradation showed a significant decline in tib-
ial nerves, which was inversely correlated to the level of NF-L in
tibial nerves. Thus, the results suggest that the elevation of NFs
content induced by PMSF could be ascribed to the inhibition of NFs
degradation.
Taken together, we previously reported the exposure to TOCP
resulted in an elevated NF degradation in peripheral nerves of
hens. In the present investigation, we extend these observations
by TOCP-intoxicated hen models pretreated with PMSF. Adminis-
tration of PMSF 24 h before TOCP exposure not only blocked the
onset and development of clinic signs of TOCP-treated hens, but
also blocked the NF degradation in peripheral nerves of hens. The
findings reported in these studies provide compelling evidence
for the implication of NF degradation in TOCP delayed neuropa-
thy. Namely, the disruption of NF homeostasis in peripheral nerves
might be involved in the onset and development of TOCP-induced
delayed neuropathy. Furthermore, our findings also suggest the
activation of proteolytic enzymes by TOCP might play a key role in
262 F. Song et al. / Toxicology 262 (2009) 258–264

Fig. 3. Time-course alteration of NF-L degradation in tibial nerve of hens pretreated with PMSF 24 h earlier and subsequently TOCP. Representative immunoblots of NF-L are
also shown below the graph. (A) and (B) represent the profile of NF-L in supernatant and pellet fractions of hen tibial nerves. NF-L was demonstrated as about 70-kDa band,
breakdown products of NF-L were around between 40 and 60 kDa. The quantity of NF-L degradation products on immunoblots from supernatant and pellet fraction of tibial
nerve was expressed as percentage of the total amount of NF-L degradation and remaining NF-L (=100%). The degradation rates of NF-L were significantly elevated in tibial
nerve of TOCP-treated hen compared to the control. Data are mean ± S.D. (bars) values (n = 6).

the mechanism of OPIDN, and the inhibition of protease-mediated
NF degradation might be a protective mechanism of PMSF against
OPIDN.
The possible role of protease in OPIDN has been men-
tioned in several previous studies. In the studies of diisopropyl
phosphorofluoridate (DFP)-induced delayed neuropathy, increased
degradation/degeneration of cytoskeletal proteins have been
observed, which suggested that the activity of neuronal proteases
could be a possible target of organophosphorus ester inducing
delayed neuropathy (Seifert and Casida, 1982; Tanaka et al., 1990).
So far, however, the role of proteolytic pathways in OPIDN has not
been studied thoroughly. This study provides further supports for
the relationship between the activation of proteolytic enzymes and
OPIDN, although the specific proteolytic pathways of NF and regu-
lating mechanisms are still unknown.
NFs are normally degraded by proteases at the nerve terminal,
and degraded material is normally transported back to the cell body
for further processing. In this regard, it is interesting that alter-
ations of calcium-activated neutral protease (calpain) have been
reported in OPIDN. Calpains, a family of neutral cytosolic Ca2+ -
dependent cysteine proteases, are known to hydrolyse cytoskeletal
proteins including NFs (Perrin and Huttenlocher, 2002; Suzuki
et al., 1995). In El-Fawal’s studies, OP administration caused an
increase in calcium-activated neutral protease activity (CANP) in
brain of hens within 4 days and in sciatic nerve of hens within
2 days of administration before onset of clinical signs (El-Fawal
et al., 1990). In contrast, another study reported DFP decreased
calpain activity in hen ischiatic nerves even though there was
increased cytoskeletal degradation. In that study, Gupta concluded
that degradation of cytoskeletal proteins in OPIDN may occur
through release of intracellular calcium into the axoplasm, increase
in some other proteinases activity, or increase in the susceptibil-
ity of cytoskeletal proteins to proteinases (Gupta and Abou-Donia,
1995). As a serine protease inhibitor, PMSF cannot effectively
inhibit the activity of calpains (Shoshan-Barmatz et al., 1994).
However, the results described in this study supported the pre-
treatment of PMSF could significantly inhibit the degradation of
NF, which suggested that the activation of calpain was not a
common response in OPIDN, and there existed other proteolytic
pathways for NF degradation rather than calpain in peripheral
nerves.
There have been many reports suggesting that NF can be
degraded by the autophagy–lysosome pathway, a major route of
protein complexes and organelle clearance in eukaryotic cells.
Recent studies have highlighted the importance of basal autophagy
in intracellular quality control, particularly in the nerve tissue
and in other tissues where the cells do not divide after dif-
ferentiation. Autophagosome-like vacuoles have been shown to
be present in the degenerating axons associated with a range
of chronic neurodegenerative conditions, including Alzheimer’s,
Huntington’s diseases and their animal models, and the dysfunction
of autophagy–lysosome pathway is implicated in the pathogen-
esis of many neurodegenerative diseases (Levine and Kroemer,
2008).
Previous morphological studies on OPIDN have consistently
shown the presence of large numbers of autophagosome-like vac-
uoles in axonal dystrophic swellings of injured neurons. Although
a basal level of autophagy can play a critical role in axonal protec-
tion and altered autophagy could serve as an adaptive response
for remodeling the axon terminals for regeneration, we cannot
exclude the possibility that up-regulation of autophagy in dys-
trophic axons is actually destructive, causing overdegradation of
axonal structures. The substrates of autophagy–lysosome pathway
include cytoskeletal components such as NF. In a tissue culture
 F. Song et al. / Toxicology 262 (2009) 258–264
study, vesicular bodies rapidly transported back to the cell body
were seen in the processes of sensory neurons (Suzuki et al.,
1988). These were thought to be prelysosomal vesicles and some
could be stained with antibodies to NF triplet proteins. In line
with this, cathepsin B and D, two widely expressed lysosome pro-
teases, had also been shown to degrade NF in vitro, producing a
variety of uncharacterized proteolytic fragments (Hollenbeck and
Bray, 1987; Banay-Schwartz et al., 1987; Posmantur et al., 1998).
Furthermore, a possible involvement of the ubiquitin–proteasome
system (UPS) in NF degradation was reported in Zhai’s study
(Zhai et al., 2003). Inhibiting UPS activity by both pharmacolog-
ical and genetic means profoundly delayed axon degeneration
both in vitro and in vivo, and profoundly retarded the cleavage of
NFs.
In fact, autophagy, UPS, and cytosolic proteases such as calpains
exist within cells. It is not surprising that these degradation systems
function simultaneously and sometimes cooperate (Mizushima,
2007). For example, when calpain and proteasomal degradation are
blocked, autophagy will be activated. In contrast, the inhibition of
autophagy can induce the activation of calpain pathway (Williams
et al., 2008; Ding et al., 2007; Zhang et al., 2009). We speculate that
dysfunction of proteolytic pathway has been implicated in the ini-
tiating events occurring during onset and development of OPIDN.
A comprehensive analysis of three proteolytic pathways above-
mentioned may contribute to clarify the underlying mechanism
of NF degradation in OPIDN, and provide new ideas for exploring
the mechanisms of OPIDN. In this respect, further study remains
conducted.
In summary, in the present investigation, we have examined
the effect of pretreatment with PMSF on NFs degradation in the
peripheral nerve of hens exposed to TOCP. The results demon-
strated that pretreatment of PMSF could inhibit the NF degradation
induced by TOCP, which was likely associated with the protecting
effect of PMSF. These findings suggested the activation of prote-
olytic pathway might be implicated in the onset and development
of TOCP-induced delayed neurotoxicity. The exact role and molec-
ular mechanism of proteolytic pathway in OPIDN remains to be
elucidated.
Conflict of interest
None declared.
Acknowledgements
This work was supported by grants from Ministry of Science and
Technology of China (No. 2002CB512907), Department of Science
and Technology of Shandong Province, China (No. 2005GGC03151),
and China Postdoctoral Science Foundation (No. 20080431191).
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