Toxicology 252 (2008) 56–63
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Toxicology
journal homepage: www.elsevier.com/locate/toxicol
The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not
disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens
Wei-Yuan Hou a,b , Ding-Xin Long a , Hui-Ping Wang a , Qi Wang 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,
Datunlu Road, Beijing 100101, PR China
b
Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e
i n f o
Article history:
Received 7 June 2008
Received in revised form 25 July 2008
Accepted 25 July 2008
Available online 5 August 2008
Keywords:
Organophosphate
Delayed neurotoxicity
Neuropathy target esterase
Hen
Homeostasis
Lysophosphatidylcholine
Phosphatidylcholine
a b s t r a c t
Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity
(OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the
homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might
be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN
because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as
lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay
for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer
with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE,
LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme
in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities
of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSFtreated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient
in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the
NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed,
while the GPC level was significantly decreased. However, there is no relationship found between the GPC
level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis
disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted
after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and
the GPC level was remarkably decreased.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Organophosphorous compounds (OP) are the principal class of
insecticide and chemical warfare agents (Casida and Quistad, 1998;
Marrs et al., 1996). Certain OPs such as TOCP, which is used in
industry mainly as an additive to lubricating oil and a softener in
the manufacture of plastic products, are able to induce a delayed
neurodegenerative condition known as OP-induced delayed neurotoxicity (OPIDN) (Johnson, 1974; Craig and Barth, 1999; Winder and
Balouet, 2002). OPIDN is an axonopathy which is characterized by
distal degeneration of some long and large-diameter axons in the
peripheral nerves and the spinal cord (Lotti, 1992; Johnson, 1993).
The putative molecular target of OPIDN is a neural protein with
esterasic activity called neuropathy target esterase (NTE), which
∗ Corresponding author. Tel.: +86 10 64807251; fax: +86 10 64807099.
E-mail address: [email protected] (Y.-J. Wu).
0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2008.07.061
is intimately associated with the cytoplasmic face of the endoplasmic reticulum (Li et al., 2003; Akassoglou et al., 2004). NTE is
defined as the paraoxon-resistant and mipafox-sensitive esterase
with phenyl valerate-hydrolyzing activity and the NTE assay is fully
validated for toxicological relevance to OPIDN (Johnson, 1969, 1975,
1977). In sensitive species, OPIDN is initiated when >70% of the
NTE is inhibited by neuropathic OPs and the clinical expression
occurs 2–3 weeks later (Johnson, 1969). An aging reaction is also
required for OPIDN (Johnson, 1990). Although OPIDN has been the
subject of intense investigation for years, a definitive causal link
between OP-modified NTE and nerve damage has not yet been
established (Glynn, 2003; Read et al., 2007). Nevertheless, significant progresses have been achieved by studying the biochemical
function of NTE. The purified recombinant catalytic domain of NTE
has sequence similarity in the active site region to that of the
calcium-independent phospholipase A2 , known to have lysophospholipase (LysoPLA) activity, and displays potent LysoPLA activity
in vitro (van Tienhoven et al., 2002). Therefore, NTE may act as a
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
57
Together with the critical role of PC and LPC in the nerve system
and the biochemical function of NTE, as a LysoPLA or PLB, disruption of PC and LPC homeostasis may be a mechanism for OPIDN. To
confirm this hypothesis, we have induced OPIDN in hens with tri-ocresyl phosphate (TOCP), an inhibitor of NTE and investigated the
relationship between alteration of NTE-LysoPLA or PLA and NTEPLB or PLB activities and the alteration of the homeostasis of the
PC, LPC, and GPC.
2. Materials and methods
2.1. Reagents
Fig. 1. The pathway of neuropathy target esterase (NTE) participating in phosphatidylcholine (PC) metabolism. Choline (Cho) is transported into cells and
then phosphorylated by choline kinase (CK). Phosphocholine (P-Cho) reacts with
cytidine triphosphate (CTP) in the pathway’s rate-limiting step catalyzed by
CTP:phosphocholine cytidylyltransferase (CCT) forming CDP-choline (CDP-Cho).
CDP-Cho reacts with diacylglycerol (DAG) catalyzed by choline phosphotransferase
(CPT) to form membrane-associated PC. PC can be hydrolyzed by phospholipase
D, forming phosphatidic acid and Cho, PC can also be deacylated by NTE at the
cytoplasmic face of the endoplasmic reticulum to form soluble products: free fatty
acids and glycerophosphocholine (GPC). In addition, phospholipase A and NTE can
sequentially deacylate PC to GPC. GPC is hydrolyzed by glycerophosphorylcholine
phosphodiesterase (GPCP), forming Cho and glycerophosphate. Cho can be phosphorylated or secreted from the cell. The above information is combined from Quistad
et al. (2003), Anfuso et al. (2003), Zaccheo et al. (2004), and Read et al. (2007).
kind of LysoPLA with lysophosphatidylcholine (LPC) as its physiological substrate in mouse (Quistad et al., 2003). Further study
demonstrated that NTE also contains phospholipase B (PLB) activity
which can convert phosphatidylcholine (PC) to glycerophosphocholine (GPC) in yeast and mammalian cells (Zaccheo et al., 2004).
Collectively, these discoveries indicate that inhibition and aging of
NTE during OPIDN may affect the homeostasis of PC and LPC (see
Fig. 1 for the pathway of NTE participating in PC metabolism).
PC is the major phospholipid of eukaryotic cells, representing
about 50% of the membrane phospholipids in animal cells (Cui and
Houweling, 2002). Regulation of the PC biosynthesis, degradation,
and relative distribution among different membranous structures
is critical for cellular function (Fernández-Murray and McMaster,
2005). Inhibition synthesis or excessive breakdown of the PC has
been associated with growth arrest and apoptotic cell death (Tercé
et al., 1994; Cui et al., 1996; Anthony et al., 1999). Disruption of the
PC homeostasis happens in many neurodegenerative disorders. For
examples, breakdown of the PC has been observed in senile dementia while elevated PC levels can be found in specific brain regions
of Alzheimer’s patients (Klein, 2000; Soderberg et al., 1992). The PC
and its related choline compounds have a neuronal-specific function, in that they have been shown to promote the synthesis and
transmission of neurotransmitters (McDaniel et al., 2003). The LPC
is another important phospholipid molecule in mammalian tissues.
An elevated LPC level can induce neuronal sheath demyelination,
together with a variable degree of axonal degeneration (Hall, 1972;
Jean et al., 2002), which is similar to the pathological changes of
OPIDN, suggesting inhibition of NTE may lead to demyelination.
Myelin sheaths are extensions of the plasma membrane of Schwann
cells in the peripheral nervous system, and of oligodendrocytes in
the central nervous system. The myelin sheaths are specialized and
unique membranes that have a high content of phospholipids and
a relatively simple protein composition (Vance et al., 2000) and
therefore correct composition of phospholipids is important for
maintaining its proper function.
TOCP (purity >99%) was purchased from BDH Chemicals Co. Ltd. (Poole, England). Coomassie brilliant blue G-250 and phenylmethylsulfonyl fluoride (PMSF)
were purchased from Fluka Chemika (Buchs, Switzerland). Mipafox and phenyl
valerate were synthesized in our laboratory as described previously (Johnson, 1982).
Atropine sulfate was obtained from Minsheng Pharmaceutical Factory (Hangzhou,
China). Benzenesulfonyl fluoride, paraoxon, sn-glycero-3-phosphocholine phosphodiesterase, choline oxidase, peroxidase (horseradish), PC, LPC, and GPC
were purchased from Sigma (St. Louis, MO, USA). 3-(N-Ethyl-3-methylanilino)-2hydroxypropanesulfonic acid sodium salt (TOOS) was obtained from Nanjing Robiot
Company (Nanjing, China). 4-Aminoantipyrine was obtained from Beijing Xizhong
Chemical Factory (Beijing, China) and silica gel 60 F254 plates (20 × 20, 0.25-mm
thick) were purchased from Merck (Darmstadt, Germany).
2.2. Animals
Adult Beijing white laying hens (8 months old and 1.5 kg in size) used in this
study were purchased from the Dabei Poultry Farm (Beijing, China). They were
housed in cages individually. The birds were acclimatized for at least 1 week prior
to the start of the experiment. During the experiment periods, the temperature in
the hen house was maintained at 22 ◦ C and 50% humidity with a light/dark cycle of
12 h.
2.3. Administration
Thirty hens were divided into three groups (Control, TOCP-, and PMSF-treated)
with 12 hens in each experimental group and 6 hens in the control group. Hens in
the TOCP group were orally given a single dose of 750 mg/kg in a gelatin capsule
while three hens in control group were given an empty gelatin capsule. The hens
in the PMSF group were injected with PMSF (100 mg/kg, sc) dissolved in dimethyl
sulfoxide (DMSO), while the other three hens in the control group received DMSO
(0.8 mg/kg, sc) only. From the first day after exposure to the chemicals, the hens were
examined daily (twice per day, in the morning and late afternoon) for the delayed
neurotoxic signs until the 14th day. On completion of 4 h, 4 days, and 14 days postdosing, four hens from chemical-treated groups at each time point and the hens
from control group at the last time point were sacrificed, respectively, by cervical
decapitation. The whole brain, spinal cord, and sciatic nerve were quickly dissected
and frozen in liquid nitrogen before storing at −80 ◦ C.
2.4. NTE activity assay
Nervous tissues were homogenized in TE buffer (50 mM Tris–HCl, 0.2 mM EDTA,
pH 8.0) and centrifuged at 100 × g at 4 ◦ C for 2 min. NTE activity in the supernatant fraction was determined from colorimetric assay of the phenol formed by the
absorbance difference for phenyl valerate hydrolysis between samples exposed to
40 ␮M paraoxon and those with both 40 ␮M paraoxon plus 50 ␮M mipafox according
to Johnson (1977) with modification for reduced volume microassay as previously
described in our lab and expressed as nanomoles of phenol formed per minute per
milligram of protein with phenol as the standard (Chang et al., 2006). Concentration
of protein was measured by the method of Bradford using bovine serum albumin as
standard (Bradford, 1976).
2.5. NTE enzyme aging assay
The aging measurement procedure was previously described by Kellner et al.
(2000) with modifications for reduced volume. Nerve tissue samples were homogenized in 10% (w/v) TE buffer. A fresh solution of potassium chloride (KCl) and
potassium fluoride (KF) reactivation buffer was prepared (250 mM in 50 mM Triscitrate, 0.2 mM EDTA, pH 5.2); and reactivation was initiated by adding 0.25 ml
nerve tissue homogenate to 1.5 ml KF reactivation buffer. Another 0.25 ml aliquot
was added to 1.5 ml KCl-containing buffer in plastic test tubes (buffer prewarmed to
37 ◦ C in shaking water bath). After 30 min incubation at 37 ◦ C, tubes were cooled
on ice and 4.25 ml ice-cold distilled water was added to each to slow the reaction. The cooled tubes were centrifuged at 27,000 × g for 60 min, the supernatant
was discarded, and the pellet was resuspended in 1 ml TE buffer. NTE activity was
58
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
determined as described above except using benzenesulfonyl fluoride (250 ␮M, final
concentration) instead of paraoxon (Johnson et al., 1985).
2.6. NTE-LysoPLA, NTE-PLB, total LysoPLA and PLB activities assay
The procedures to measure NTE-LysoPLA and total LysoPLA were modified from
a method for analysis of LPC in human plasma as described by Quistad et al.
(2003) and expressed as mAU/min (Kishimoto et al., 2002; Quistad et al., 2003).
NTE-LysoPLA was defined as the paraoxon-resistant and mipafox-sensitive LPChydrolyzing activity. Briefly, two reagents (A and B) were used. Reagent A contains
3-(N-ethyl-3-methylanilino)-2-hydroxysulfonate (3 mM), peroxidase (10 units/ml),
sn-glycero-3-phosphocholine phosphodiesterase (0.1 units/ml), and choline oxidase
(10 units/ml). Reagent B contains 5 mM 4-aminoantipyrine. All reactants were dissolved in 100 mM Tris buffer (pH 8.0) containing 1 mM calcium chloride and 0.01%
Triton X-100. The supernatant fraction (700 × g, 10 min, 4 ◦ C) of nervous tissues
homogenate (nervous tissues were homogenized in 20% (w/v) TE buffer) was used
as the enzyme samples. Four reaction groups (I–IV) with six replicates each were
designed. The total reaction volume of each group is 320 ␮l. 120 ␮l reagents A,
80 ␮l reagent B, and 15 ␮l samples were added to individual wells containing Tris
buffer in a 96-well polystyrene plate. Then 40 ␮M (final concentration) paraoxon
and 50 ␮M (final concentration) mipafox plus paraoxon were added, respectively, in
groups III and IV. After a 20-min incubation at 25 ◦ C, LPC (250 ␮M, final concentration) was introduced in all groups except Tris buffer added instead in group I. The
enzyme activity was measured by kinetic assay of absorbance at 570 nm for 10 min
at 25 ◦ C using a microplate reader with group I as the blank. Total LysoPLA was measured from group II. The difference of LPC-hydrolysis activity between groups III
and IV is the NTE-LysoPLA activity as the definition. The NTE-PLB and total PLB were
determined with the same procedure except that the substrate was PC instead of
LPC.
concentrations of inorganic phosphorus were calculated using a standard phosphorus curve. The amount of each phospholipid and choline metabolites was
given using the amount of recovered phosphate in each spot and the results were
expressed as of nanomoles or micromoles of phosphorus per gram nerve tissue wet
weight.
2.10. Statistical analysis
Data were generally expressed as mean ± standard error values and groups of
data were compared by ANOVA. The differences of decrease in GPC levels caused
by TOCP and PMSF were analyzed by three-way ANOVA to examine interactions
among drug, tissue, and time, and then paired-groups t-test was applied to analyze
the differences in each nerve tissue. A difference between means was considered
significant at p < 0.05. Correlation analysis between NTE and NTE-LysoPLA, NTE and
NTE-PLB were made by linear correlation using SPSS 13.0.
3. Results
3.1. TOCP can induce complete OPIDN in hens
We induced OPIDN in hens using TOCP with PMSF as a negative control. Signs of delayed neuropathy (progressive ataxia) were
first observed on the 8th day post-dosing in hens of the TOCP
group, progressing into paralysis by the 14th day for all hens. However, no symptom of delayed neuropathy was observed in hens
of all other groups during the whole experiment period (data not
shown).
2.7. Lipids and choline metabolites extraction
Lipids and choline metabolites were extracted from nervous tissues with a
chloroform/methanol mixture (2:1, v/v) using the procedure of Folch et al. (1957)
with some modifications. Briefly, tissue samples were weighted, and then homogenized in 10% (w/v) TE buffer at 4 ◦ C. The homogenate was centrifuged at 3000 rpm
for 10 min, then the supernatant (S1 ) and the sediment (P1 ) was separated. Fivefold the S1 volume of chloroform/methanol mixture (2:1, v/v) was introduced
and stirred vigorously, and then the upper aqueous phase (U1 ) and the lower
organic phase (L1 ) were recovered after centrifugation (1000 rpm, 5 min). The P1
was homogenized again in 20-fold volume chloroform/methanol mixture (2:1,
v/v) of the tissue sample weight at 4 ◦ C, and then centrifuged at 3000 rpm for
10 min. The supernatant (S2 ) and the sediment (P2 ) were separated. The S2 was
recovered. The P2 was re-extracted in 10-fold volume chloroform/methanol mixture (2:1, v/v) of the tissue sample weight and supernatant (S3 ) was recovered
after centrifugation (3000 rpm, 10 min). S2 and S3 were combined, and then 0.1 M
KCl (0.2-fold volume of the combined S2 and S3 ) was added and stirred vigorously. The upper aqueous phase (U2 ) and lower organic phase (L2 ) were recovered
after centrifugation (1000 rpm, 5 min). Then U1 and U2 , L1 and L2 were combined, respectively. Organic extracts were evaporated until dry under nitrogen
stream. Water soluble choline metabolites were evaporated under nitrogen stream
to evaporate methanol, then freeze-dried. After evaporation, the lipid extract and
choline metabolites were redissolved in a small volume of chloroform/methanol
(1/1, v/v) and methanol/water (1/1, v/v), respectively, for thin-layer chromatography
(TLC).
2.8. Separation of phospholipids and choline metabolites by TLC
Analysis of phospholipids was achieved by one-dimensional TLC on silica gel
60 plates using chloroform/methanol/acetic acid/acetone/water (40/25/7/4/2, v/v)
as the solvent according to the method of Wang and Gustafson (1992). The choline
metabolites were fractionated by TLC in methanol/0.6% NaCl/25% aqueous ammonia
(10/10/1, v/v) (Williams and McMaster, 1998). The individual phospholipids and
choline metabolites were visualized on TLC plates by staining with iodine vapor.
The bands corresponding to PC, LPC, and GPC were scraped into glass tubes for
quantification.
2.9. Measurement of inorganic phosphorus
Inorganic phosphorus in each phospholipid and choline metabolites fraction
was measured using the method of Vaskovsky et al. (1975) with some modifications. Briefly, 0.2 ml 72% perchloric acid was added to the scrapings, and
the tubes were digested for 20 min at 190 ◦ C in an electrically heated metal
block, then the temperature was elevated to 220 ◦ C to allow the perchloric acid
to evaporate completely. After cooling, 0.3 ml water and 2.7 ml working solution were added and mixed thoroughly with a vibrator mixer, then heated
in a boiling water bath for 5 min. After centrifugation (1500 rpm, 5 min), the
absorbance of the supernatant at 815 nm was measured against a blank. The
3.2. TOCP and PMSF inhibit NTE, NTE-LysoPLA, and NTE-PLB
activities in hens
To examine the effect of TOCP and PMSF on the NTE, NTELysoPLA, and NTE-PLB activities in vivo, crude extracts were
prepared from the brain, spinal cord, and sciatic nerve of the hens
treated with TOCP, PMSF and controls for 4 h, 4 days, and 14 days
respectively, and the NTE, NTE-LysoPLA and NTE-PLB activities were
measured and compared. As shown in Table 1, both TOCP and PMSF
significantly inhibited the NTE, NTE-LysoPLA, and NTE-PLB activities at the three chosen time points in all three nervous tissues.
Remarkably, the inhibition of NTE and NTE-LysoPLA at 4 h, 4 days,
and 14 days after TOCP and PMSF exposure in brain, spinal cord,
and sciatic nerve showed high correlation with the R2 of 0.93,
0.92, and 0.79, respectively. Similarly, the inhibition of NTE and
NTE-PLB in the three nervous tissues by the two toxicants also
showed correlation with the R2 of 0.77, 0.85, and 0.73, respectively.
Although both TOCP and PMSF could inhibit the activities of the
NTE, NTE-LysoPLA, and NTE-PLB, the dynamics of the inhibition
were different between TOCP and PMSF (Table 1). In TOCP-treated
groups, the peak inhibition was observed at 4 days after administration. The inhibition of these enzymes by PMSF was much faster
than that of TOCP with the peak inhibition occurred at 4 h after
dosing. Furthermore, the inhibition of NTE, NTE-LysoPLA, and NTEPLB by TOCP or PMSF showed a similar trend in all three nervous
tissues.
3.3. TOCP induces NTE aging in vivo
It is known that TOCP can inhibit and age NTE, while PMSF cannot age NTE, though it is a potent NTE inhibitor. To confirm this
observation, the aging of NTE was measured in the brain, spinal
cord, and sciatic nerve at 4 h, 4 days, and 14 days after exposure to
TOCP and PMSF. As shown in Table 2, the hens given TOCP showed
that almost all inhibited NTE was of the aged type in the brain,
spinal cord, and sciatic nerve. As expected, the opposite result was
seen in hens administered with PMSF, with almost all of inhibited
NTE unaged type in all three nervous tissues.
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
59
Table 1
The activities of NTE (nmol phenol/min/mg protein), NTE-LysoPLA (mAU/min) and NTE-PLB (mAU/min) after TOCP and PMSF administration in hens’ nerve tissues
Tissue
Enzyme
4h
4 days
PMSF
TOCP
PMSF
TOCP
PMSF
Brain
NTE
NTE-LysoPLA
NTE-PLB
11.29 ± 1.34 (56)
4.14 ± 0.39 (59)
4.05 ± 0.63 (19)
2.59 ± 0.33 (90)
2.18 ± 0.31 (78)
2.71 ± 0.28 (46)
7.17 ± 0.83 (72)
3.42 ± 0.39 (66)
3.41 ± 0.37 (32)
7.55 ± 0.59 (70)
3.41 ± 0.29 (66)
3.98 ± 0.18 (20)
12.91 ± 1.32 (49)
5.26 ± 0.03 (47)
3.84 ± 0.84 (23)
13.35 ± 3.15 (48)
4.55 ± 0.45 (54)
4.27 ± 0.47 (15)
Spinal
cord
NTE
NTE-LysoPLA
NTE-PLB
4.36 ± 0.29 (49)
2.39 ± 0.15 (49)
2.48 ± 0.09 (34)
1.35 ± 0.34 (84)
0.92 ± 0.04 (80)
1.70 ± 0.26 (54)
2.44 ± 0.36 (71)
1.69 ± 0.20 (64)
2.22 ± 0.03 (41)
2.48 ± 0.32 (71)
1.82 ± 0.34 (61)
2.31 ± 0.52 (38)
4.55 ± 0.46 (47)
2.76 ± 0.14 (41)
2.83 ± 0.25 (24)
4.82 ± 0.44 (44)
2.51 ± 0.16 (47)
2.64 ± 0.10 (29)
Sciatic
nerve
NTE
NTE-LysoPLA
NTE-PLB
1.23 ± 0.08 (50)
1.17 ± 0.08 (59)
1.02 ± 0.09 (45)
0.28 ± 0.04 (89)
0.76 ± 0.02 (73)
0.27 ± 0.08 (85)
0.33 ± 0.06 (87)
1.06 ± 0.18 (63)
0.76 ± 0.19 (59)
0.83 ± 0.03 (66)
1.36 ± 0.08 (52)
0.98 ± 0.05 (47)
1.64 ± 0.05 (34)
1.58 ± 0.11 (44)
1.18 ± 0.21 (36)
1.62 ± 0.33 (34)
1.66 ± 0.40 (41)
1.11 ± 0.19 (40)
TOCP
14 days
Data were expressed as mean ± S.E. (n = 3). The numbers in parentheses represent the inhibition rates (%). The control NTE activity in brain, spinal cord, and sciatic
nerve was defined as 25.43 ± 0.35, 8.54 ± 0.46, and 2.47 ± 0.24 nmol phenol/min/mg protein, respectively, the control NTE-LysoPLA activity was 9.97 ± 0.87, 4.70 ± 0.22, and
2.83 ± 0.42 mAU/min, respectively, and the control NTE-PLB activity was 5.00 ± 0.39, 3.73 ± 0.18, and 1.85 ± 0.22 mAU/min respectively. NTE and NTE-LysoPLA or NTE-PLB
inhibition rates were analyzed by linear correlation, R2 = 0.93, p < 0.01, and R2 = 0.76, p < 0.05, respectively, in brain, R2 = 0.93, p < 0.01, and R2 = 0.85, p < 0.01, respectively, in
spinal cord and R2 = 0.79, p < 0.05 and R2 = 0.74, p < 0.05, respectively, in sciatic nerve.
3.4. TOCP and PMSF inhibit total LysoPLA and total PLB activities
in hens
To investigate the effect of TOCP and PMSF on total LysoPLA and
total PLB activities in vivo, these two enzymes activities from brain,
spinal cord, and sciatic nerve were assayed after 4 h, 4 days, and
14 days administration with single doses of TOCP and PMSF. The
activities of the total LysoPLA (Fig. 2A–C) and total PLB (Fig. 2D–F)
significantly decreased in the three nervous tissues at the three
chosen time points after dosing. The inhibition of total LysoPLA by
TOCP or PMSF exhibited similar trend in the three different nervous
tissues. However, the inhibition of this enzyme showed different
characteristic between TOCP and PMSF. In TOCP-treated groups,
the peak inhibition of total LysoPLA was observed at 4 days after
administration while the peak inhibition occurred at 4 h in PMSFtreated groups. Furthermore, PMSF was a more potent inhibitor of
total LysoPLA than TOCP. The characteristic of inhibition of total
PLB by TOCP and PMSF was similar, and both toxicants leaded to
a peak inhibition occurred at 4 days post-dosing. The inhibition of
total PLB by the two toxicants also exhibited a similar trend in the
three different nervous tissues. However, TOCP caused more of an
effect on total PLB activity at 4 days after administration in all three
nervous tissues than PMSF did, which indicates that TOCP may be
a more potent PLB inhibitor than PMSF.
3.5. TOCP and PMSF do not alter the levels of PC and LPC but
decrease the level of GPC in hens
After confirming the observations that NTE, NTE-LysoPLA, NTEPLB, total LysoPLA, and total PLB activities significantly decreased
after TOCP and PMSF exposure, we focused attention on whether
the inhibition of these enzymes could affect the levels of PC, LPC and
GPC, and whether the aged and unaged NTE had different effects.
Thus, the levels of PC, LPC, and GPC were determined in brain, spinal
cord, and sciatic nerve at 4 h, 4 days, and 14 days after TOCP and
PMSF exposure. Surprisingly, the levels of PC (Fig. 3A–C) and LPC
(Fig. 3D–F) were not significantly changed in all three nervous tissues at the three chosen time points, despite of NTE, NTE-LysoPLA,
NTE-PLB, total LysoPLA, and total PLB activities were clearly inhibited. In contrast, a remarkable decrease of GPC level (Fig. 3G–I) was
observed in brain at 4 h, 4 days, and 14 days, and spinal cord and
sciatic nerve at 4 days and 14 days after TOCP and PMSF exposure.
Moreover, the decrease of GPC level induced by TOCP and PMSF
exposure exhibited similar trend in brain, spinal cord, and sciatic
nerve. To further determine whether the decrease of GPC induced
by TOCP and PMSF was different, a three-way ANOVA was used to
look for the interactions among drug, tissue, and time. As a result,
interactions were observed among the three factors (p < 0.01), however, no significant difference was found on the decrease of GPC
caused by TOCP and PMSF in each nervous tissue by paired-groups
t-test analysis (p > 0.05). These data indicate that the decrease of
GPC level induced by TOCP and PMSF is not different in brain, spinal
cord, and sciatic nerve.
4. Discussion
Although OPIDN has been the subject of intense investigation
for years, little is known about early biochemical events during this
disorder except that inhibition and aging of NTE is required. Never-
Table 2
The aged and unaged NTE activities (nmol phenol/min/mg protein) in the nerve tissues from TOCP- and PMSF-treated hens
Tissue
NTE activity
4h
4 days
TOCP
14 days
TOCP
PMSF
PMSF
TOCP
PMSF
Brain
Inhibited
Unaged
Aged
13.89 ± 1.78
0.87 ± 0.77
13.02 ± 1.70
22.08 ± 0.79
21.92 ± 0.27
0.16 ± 0.79
17.39 ± 1.31
0.23 ± 0.17
17.16 ± 1.28
17.95 ± 1.29
17.65 ± 0.80
0.30 ± 0.95
13.32 ± 0.48
0.70 ± 0.36
12.62 ± 0.57
12.26 ± 0.44
11.88 ± 1.39
0.38 ± 0.97
Spinal
cord
Inhibited
Unaged
Aged
4.51 ± 0.16
0.42 ± 0.34
4.09 ± 0.18
7.20 ± 0.24
7.12 ± 0.73
0.08 ± 0.02
6.15 ± 0.23
0.37 ± 0.01
5.78 ± 0.22
5.89 ± 0.40
5.81 ± 0.11
0.08 ± 0.03
4.26 ± 0.29
0.44 ± 0.09
3.82 ± 0.37
3.77 ± 0.39
3.75 ± 0.64
0.02 ± 0.25
Sciatic
nerve
Inhibited
Unaged
Aged
1.18 ± 0.02
0.10 ± 0.08
1.08 ± 0.07
2.13 ± 0.07
2.07 ± 0.05
0.06 ± 0.12
2.12 ± 0.04
0.13 ± 0.11
1.99 ± 0.12
1.81 ± 0.05
1.69 ± 0.16
0.12 ± 0.21
0.83 ± 0.31
0.11 ± 0.15
0.72 ± 0.18
1.10 ± 0.27
1.01 ± 0.18
0.09 ± 0.15
Data were expressed as mean ± S.E. (n = 3). Unaged NTE = the NTE activity after treatment with KF subtracted the NTE activity after treatment with KCl; aged NTE = control NTE
activity subtract the NTE activity after treatment with KF, inhibited NTE activity = aged NTE activity + unaged NTE activity (see the details of aging measurement described in
Section 2). The control NTE activity in brain, spinal cord, and sciatic nerve were, respectively, defined as 25.43 ± 0.35, 8.54 ± 0.46, and 2.47 ± 0.24 nmol phenol/min/mg protein.
60
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
Fig. 2. The alteration of total LysoPLA activity in hens’ brain (A), spinal cord (B), and sciatic nerve (C) and total PLB activity in hens’ brain (D), spinal cord (E), and sciatic nerve
(F) after 4 h, 4 days, and 14 days administration with TOCP and PMSF. Data were presented as a percentage of control total LysoPLA or total PLB activities. The control total
LysoPLA activities in brain, spinal cord, and sciatic nerve were defined as 42.02 ± 3.58, 22.50 ± 2.38, and 16.48 ± 1.86 mAU/min, respectively. The control total PLB activities in
brain, spinal cord, and sciatic nerve were, respectively, defined as 19.73 ± 2.33, 9.95 ± 1.23, and 8.22 ± 0.42 mAU/min. Data were expressed as mean ± S.E. and were compared
by ANOVA between control and administration groups, * p < 0.05, ** p < 0.01, n = 3.
theless, the recent evidence that NTE participates in phospholipid
metabolism may provide some clues to understand OPIDN. In the
present study, we examined the PC and LPC homeostasis in OPIDN
hens with TOCP.
Adult hens have been used as animal model for experimental
studies of OPIDN due to their sensitivity and the development of clinical signs similar to those seen in humans (Schwab
and Richardson, 1986; Barrett et al., 1985). TOCP has been the
prototype-inducing agent for delayed neurotoxicity, as it can inhibit
and age NTE. PMSF does not initiate OPIDN in hens because NTE
inhibited by PMSF does not undergo aging reaction (Johnson, 1990).
However, PMSF can modify the clinical effects of both toxic and
traumatic insults (Pope and Padilla, 1990; Lotti et al., 1991). PMSF
protects the animal from OPIDN when given before the neurotoxic OPs, yet exacerbated the polyneuropathy when given after OP
administration (Johnson, 1974). Our data showed that both TOCP
and PMSF significantly inhibited NTE activity in brain, spinal cord,
and sciatic nerve of hens. The NTE inhibited by TOCP underwent
aging reaction, while the NTE inhibited by PMSF did not. Consequently, we successfully induced OPIDN model in hens using a
single dose of TOCP, while did not in the hens treated with PMSF.
Previous studies showed that the inhibition of NTE and NTELysoPLA in mice brain by delayed toxicants in vivo or in vitro exhibit
the same extent and Nte+/− transgenic mice are similarly deficient
in NTE-LysoPLA activity, providing evidence that NTE-LysoPLA and
NTE are very similar or identical (Quistad et al., 2003). Here the
inhibition of NTE-LysoPLA and NTE-PLB activities were compared
with that of NTE in hens’ brain, spinal cord, and sciatic nerve at 4 h,
4 days, and 14 days after TOCP and PMSF exposure. The inhibition
of NTE and NTE-LysoPLA at the three tested time points after TOCP
and PMSF exposure in the three nervous tissues showed high correlation. Similarly, the inhibition of NTE and NTE-PLB also showed
high correlation, though the correlation coefficients were less than
those of NTE and NTE-LysoPLA. Furthermore, the inhibition of NTE,
NTE-LysoPLA, and NTE-PLB by TOCP or PMSF in the three different
nervous tissues exhibited a similar trend. These observations indicated that the three enzymes may contain a similar characteristic
in the three different nervous tissues and NTE and NTE-LysoPLA or
NTE-PLB in hens, similar to mice, may be very similar or identical.
The data obtained in hens provided strong support for the claim
that NTE functions as LysoPLA or PLB in some extent. Interestingly,
we observed that NTE-LysoPLA activity assayed with LPC as the substrate was much higher than that of NTE-PLB assayed with PC as the
substrate in the three nervous tissues. This may be in part due to the
fact that LPC was the more avidly hydrolyzed substrate than PC for
NTE under the current experimental conditions. Previous studies
showed that the rate and selectivity of bond cleavage observed in
lipase assays in vitro are profoundly affected by the physicochemical nature of the substrate (Reynolds et al., 1991; Ackermann et
al., 1994; Lin et al., 2000; Zaccheo et al., 2004). It is possible that
in vivo NTE could deacylate PC more efficiently than that observed
in vitro experiments. In principle, the deacylation at both sn-2 and
sn-1 positions of PC could be mediated either by a single enzyme
with PLB activity or by sequential action of a phospholipase A2 and
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
61
Fig. 3. The alteration of PC level in hens’ brain (A), spinal cord (B), and sciatic nerve (C), LPC level in brain (D), spinal cord (E), and sciatic nerve (F) and GPC level in hens’ brain (G),
spinal cord (H), and sciatic nerve (I) after 4 h, 4 days, and 14 days administration with TOCP and PMSF. Data were presented as a percentage of control PC, LPC, and GPC levels in
nerve tissues. The control PC levels in brain, spinal cord, and sciatic nerve were, respectively, defined as 13.47 ± 1.23 ␮mol/g wet brain weight, 13.48 ± 1.07 ␮mol/g wet spinal
cord weight, and 4.89 ± 0.87 ␮mol/g wet sciatic nerve weight. The control LPC levels in brain, spinal cord, and sciatic nerve were, respectively, defined as 347.53 ± 5.15 nmol/g
wet brain weight, 403.31 ± 11.97 nmol/g wet spinal cord weight, and 297.83 ± 20.46 nmol/g wet sciatic nerve weight. The control GPC levels in brain, spinal cord, and sciatic
nerve were, respectively, defined as 570.86 ± 15.41 nmol/g wet brain weight, 313.10 ± 5.98 nmol/g wet spinal cord weight, and 390.01 ± 5.60 nmol/g wet sciatic nerve weight.
Data were expressed as mean ± S.E. and were compared by ANOVA between control and administration groups, * p < 0.05, ** p < 0.01, n = 3.
a LysoPLA (Zaccheo et al., 2004). Thus, it seems possible that NTE
may play the role of PLB with PC as its substrate as well as the role
of LysoPLA acting on the established LPC pool.
TOCP and PMSF had different characteristics on inhibition of
NTE, NTE-LysoPLA, total LysoPLA, and NTE-PLB activities. Inhibition
of these enzymes by PMSF was much faster than that by TOCP, and
PMSF was a more potent inhibitor of LysoPLA than TOCP. Possible
reasons may exist besides the chemical nature themselves. PMSF,
as a broad-spectrum protease inhibitor, may inhibit more LysoPLAs
than TOCP. TOCP needs a process of metabolism in vivo (saligenin
cyclic-o-tolyl phosphate, the active neurotoxic metabolite of TOCP)
to retard its toxicological effect (Suwita and Abou-Donia, 1990). The
inhibition of total LysoPLA and total PLB by TOCP or PMSF showed
a similar trend in brain, spinal cord, and sciatic nerve. This suggests
that the part of TOCP-sensitive or PMSF-sensitive of total LysoPLA
and total PLB may have a similar characteristic in the three different
nervous tissues.
Although the inhibition and subsequent aging of NTE has been
proposed to be an initiating event in OPIDN, the events that occur
between NTE inhibition and the appearance of clinical effects are
not completely understood (Glynn, 2003; Read et al., 2007). However, remarkable progress has been achieved with the discovery
that NTE is a putative phospholipase. This discovery provides new
clues to possible cellular functions of NTE; disruption of PC and
LPC homeostasis may be a mechanism for OPIDN. However, no significant alterations of PC and LPC levels were observed in brain,
spinal cord, and sciatic nerve at different time points after TOCP
and PMSF exposure in the present study, though the TOCP-treated
hens showed typical signs of OPIDN and the key metabolic enzymes
for PC and LPC were clearly inhibited. This suggested that TOCP and
PMSF administration did not disrupt PC and LPC homeostasis, and
the alteration of PC and LPC homeostasis may not be a mechanism
involved in OPIDN by neuropathic OPs. However, the level of GPC,
the product of PC or LPC, remarkably decreased and both TOCP and
PMSF administration showed similar trends in the three nervous
tissues, which indicated that alteration of GPC level by aged and
unaged of inhibited NTE was not different, and the alteration of
GPC level was not directly related with OPIDN. The inhibition of
total LysoPLA, NTE-LysoPLA, total PLB, and NTE-PLB did not increase
the PC and LPC levels, suggesting feedback mechanisms may exist
62
W.-Y. Hou et al. / Toxicology 252 (2008) 56–63
in vivo. The hens’ bodies may maintain the homeostasis of PC and
LPC by reducing their synthesis or activating other degradation
pathways such as PC-specific phospholipase C or phospholipase D
(Adibhatla et al., 2006). GPC is mainly produced by deacylation of
PLB on PC or LysoPLA on LPC, and the inhibition of PLB or LysoPLA could possibly result in the decrease of GPC level. Drosophila
with mutations in the gene for the NTE homologue, Swiss cheese
protein, PC level in the head are significantly increased compared
to wild-type and show age-dependent degeneration in the adult
(Kretzschmar et al., 1997; Muhlig-Versen et al., 2005). However,
no significant alteration of PC level was observed in hens when
NTE was clearly inhibited. This result suggested that Drosophila may
require the activity of SWS to maintain PC homeostasis, while vertebrate may have more NTE-related phospholipases involving in
maintaining PC homeostasis.
It is well-known that serine hydrolases are the potential OPsensitive targets that exist in organisms (Casida and Quistad, 2005;
Vose et al., 2007). There are several hundred serine hydrolases
in humans and more than 600 in Drosophila based on genomic
evidence (Yousef et al., 2003; Casida and Quistad, 2005). Each serine hydrolase has a specific function and every OP has a unique
inhibitory profile. NTE is one of the long lists of serine hydrolases.
Thus, it is possible that OPIDN may be associated with the conjunct
inhibition of serine hydrolases including NTE. Unfortunately, only
a few of serine hydrolases are studied thoroughly and it is not clear
if they play a role in OP toxicology. Therefore, clarifying the potential serine hydrolases targets and their functions may provide some
clues to understanding of the mechanisms of OPIDN.
In summary, the mechanism of OPIDN was firstly investigated
from the new insight of PC and LPC homeostasis. This study
could contribute to better understand the pathogenesis involved
in OPIDN.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the grant from the National Nature
Science Foundation of China (30470228) and partly by the grants
from National 863 Program (2006AA06Z423) and the National Key
Technologies R&D Program (2006BAK02A02). We thank Dr. PingAn Chang and Ms. Jill Wentzell for assistance of the manuscript
preparation.
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