Toxicology and Applied Pharmacology 256 (2011) 330–336

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Toxicology and Applied Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon

on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells
J. Flaskos a,⁎, E. Nikolaidis a, W. Harris b, M. Sachana a, A.J. Hargreaves b,⁎
a
Laboratory of Biochemistry and Toxicology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b
School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK

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

Article history: Previous work in our laboratory has shown that sub-lethal concentrations (1–10 μM) of chlorpyrifos (CPF),
Received 5 April 2011 diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse
Revised 1 June 2011 N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton
Accepted 3 June 2011
and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon
Available online 17 June 2011
(CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions
Keywords:
similar to our previous work, sub-lethal concentrations (1–10 μM) of CPO were found to inhibit N2a cell
Organophosphorothionate pesticide differentiation. However, unlike previous studies with DZ and DZO, there was a high level of sustained
Chlorpyrifos inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also
Chlorpyrifos oxon associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the
Neurite outgrowth distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to
N2a neuroblastoma previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken
Cell differentiation together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating
Developmental neurotoxicity
N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and
Neurofilament heavy chain
maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly,
Neurofilament phosphorylation
GAP-43 further work will help to determine whether the persistent inhibition of AChE by CPO can account for the
Acetylcholinesterase different effects induced by CPO and DZO on the levels of total and phosphorylated NFH.
© 2011 Elsevier Inc. All rights reserved.

Introduction more strongly than the parent compounds (Chambers, 1992;

Organophosphorus esters (OPs) are known to cause acute toxicity
in adults, which is mainly due to the inhibition of neuronal
acetylcholinesterase (AChE). However, a growing body of epidemio-
logical and experimental evidence indicates that these compounds
may be also capable of inducing developmental neurotoxicity (Flaskos
and Sachana, 2010; Grandjean and Landrigan, 2006). Experimental
studies demonstrate that prenatal or postnatal exposure of rodents to
the organophosphorothionate compounds chlorpyrifos (CPF) and
diazinon (DZ), which are two of the most extensively used OP
pesticides worldwide, causes considerable changes in a range of
biochemical and morphological/cellular parameters related to ner-
vous system development, leading to persistent defects in behaviour
and cognition (Ricceri et al., 2006; Roegge et al., 2008; Slotkin, 2006;
Timofeeva et al., 2008).
These OPs are metabolically converted, mainly in the liver, to their
oxygen (oxon) analogues chlorpyrifos oxon (CPO; Fig. 1) and diazinon
oxon (DZO), which inhibit AChE activity up to 3 orders of magnitude
⁎ Corresponding authors.
E-mail addresses: flaskos@vet.auth.gr (J. Flaskos), alan.hargreaves@ntu.ac.uk
(A.J. Hargreaves).
Monnet-Tschudi et al., 2000). Thus, they are deemed to be responsible
for the acute toxicity following organophosphorothionate insecticide
poisoning. On the other hand, the role of these metabolites in the
developmental neurotoxicity of CPF and DZ is unclear, due to a lack of
appropriate in vivo studies involving direct administration of these
compounds to animals at sublethal doses.
However, an emerging set of in vitro data from studies using a
number of cell culture models indicates that CPO and DZO may be able
to interfere with the development of the nervous system. For example,
CPO and DZO affect glial cell development in primary cultures and cell
lines; CPO also perturbs the expression of astrocyte- and oligoden-
drocyte-specific markers at various stages of development in cell
culture aggregates of foetal rat telencephalon (Monnet-Tschudi et al.,
2000). Furthermore, CPO inhibits DNA synthesis in rat C6 glioma and
human 1321N1 astrocytoma cell lines (Guizzetti et al., 2005; Qiao
et al., 2001). Apart from its effects on C6 cell replication, CPO also
interferes with C6 cell differentiation by inhibiting the development of
extensions from these cells under differentiation-promoting condi-
tions and affecting the integrity of the microtubule network and the
levels of microtubule proteins (Sachana et al., 2008). Similar
morphological and biochemical effects on differentiating C6 cells are
also induced by DZO (Sidiropoulou et al., 2009a).

0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2011.06.002
 J. Flaskos et al. / Toxicology and Applied Pharmacology 256 (2011) 330–336
Fig. 1. Chemical structure of chlorpyrifos oxon. Shown is a schematic diagram of the
chemical structure of CPO. The asterisk indicates that the oxygen atom shown arises
from the bioactivation of the parent compound CPF, which has a sulphur atom in this
position, by specific microsomal cytochrome P450 enzymes.
With respect to in vitro effects of oxon metabolites on the
development of neuronal cells, CPO has been shown to affect the
expression of neuronal-specific markers in brain cell aggregate
cultures (Monnet-Tschudi et al., 2000) and the expression of
developmentally relevant transcription factors in cultures of embry-
onic rat cortical and hippocampal neurons (Schuh et al., 2002).
However, data from morphological studies are not clear as to whether
CPO can readily interfere with the process of axonal/neurite
outgrowth. Thus, in the rat PC12 cell line, CPO has no effect during
the initiation phase of neurite outgrowth up to 24 h nor on the
elaboration phase from 48 to 96 h and an inhibitory effect is only noted
following a 7-day cell exposure to CPO (Das and Barone, 1999).
Moreover, in primary cultures of embryonic rat superior cervical
(Howard et al., 2005) and dorsal root (Yang et al., 2008) ganglia
neurons, CPO influences axonal length but has no effect on the number
of axons extended.
In this study, we have assessed the capacity of CPO to interfere with
the outgrowth of axon-like neurites in differentiating mouse N2a
neuroblastoma cells. Using this cell culture system we have recently
found that DZO induces rapid and potent inhibitory effects on neurite
outgrowth; following 24 h exposure to a sub-lethal concentration of
10 μM DZO, the number of long axon-like processes was reduced by
more than 80% compared to the non-DZO-treated control (Sidiropoulou
et al., 2009b). In addition, in the present study we have investigated the
effects of CPO on cytoskeletal and axon growth-associated proteins,
which have been found to be affected in N2a cells after treatment with
10 μM DZO. Throughout the study, we have employed CPO at non-
cytotoxic concentrations of up to 10 μM, using the same N2a cell growth
and differentiation conditions used previously to allow valid compar-
isons to be made between CPO and DZO and also between CPO and the
parent compound CPF.
Materials and methods
Reagents and materials
Cell culture reagents and materials were purchased from Scientific
Laboratory Supplies (SLS, Wilford, UK) or BioWhittaker UK Ltd
(Wokingham, UK), with the exception of dibutyryl cAMP, which was
obtained from Sigma Aldrich Co Ltd (Sigma: Poole, UK). Mouse
monoclonal antibodies against GAP-43 (clone GAP-7B10), α-tubulin
(clone B512), and neurofilament heavy chain (NFH: clone N52) were
obtained from Sigma. Monoclonal anti-phosphorylated NFH (pNFH)
antibody SMI31 and Ta51 were supplied by Cambridge Bioscience
(Cambridge, UK). Secondary antibodies conjugated with either
fluorescein isothiocyanate (FITC) or horseradish peroxidase (HRP)
331
were purchased from DakoCytomation (Ely, UK). Electrophoresis gel
and buffer reagents were obtained from Gene Flow (Fradley, UK) or
Sigma, whereas All Blue Precision Plus protein molecular weight
standards were purchased from BioRad Laboratories Ltd (Hemel
Hempstead, UK). Hybond-C nitrocellulose membrane filters and ECL
developing reagents were from GE Healthcare (Amersham, UK).
Chlorpyrifos oxon (purity 98.6%) was supplied by Greyhound
Chromatography (Birkenhead, UK).
Growth and maintenance of cell lines
Mouse N2a neuroblastoma cells were maintained in the logarith-
mic phase of growth at 37 °C in a humidified atmosphere of 5%
CO2/95% air. Mitotic cells were cultured in Dulbecco's modified Eagles
medium (DMEM) supplemented with 10% (v/v) foetal bovine serum
(FBS), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin
(100 μg/ml) and passaged at 60–80% confluence.
Induction of cell differentiation for measurement of neurite outgrowth
Only cells up to passage number 20 were used for differentiation
experiments. Cell differentiation was induced by serum withdrawal and
the addition of dibutyryl cAMP (Flaskos et al., 2007; Hargreaves et al.,
2006; Harris et al., 2009a, 2009b; Sidiropoulou et al., 2009b). Briefly,
mitotic cell monolayers were detached by pipette at 60–80% confluence
and harvested by centrifugation. Cell pellets were resuspended in 1 ml
growth medium and cell number determined in a haemocytometer
chamber. Cells were diluted to a density of 50,000 cells/ml before being
seeded into 24-well culture dishes with a total volume of 0.5 ml growth
medium per well.
After 24 h growth recovery, the medium was carefully removed
from each well and replaced with serum free medium (i.e. growth
medium minus FBS) containing 0.3 mM cAMP and with or without
CPO (final concentration 1–10 μM). Cells were returned to the CO2
incubator and allowed to differentiate for up to 24 h. CPO was
prepared as 200-fold concentrated stock solutions in dimethyl
sulphoxide (DMSO) and added to the serum free medium immedi-
ately before use. Control cells were treated with serum free medium
containing the same amount of DMSO (0.5% v/v).
After 24 h differentiation, serum free medium was carefully
removed by aspiration from the edge of the well and cell monolayers
were fixed at −20 °C in 90% (v/v) methanol in Tris buffered saline
(TBS: 10 mM Tris, 140 mM NaCl pH 7.4). They were then stained for
2 min in Coomassie Brilliant Blue and excess stain removed by
aspiration followed by 2 rinses in distilled water. The number of
axon-like neurites per 100 cells was estimated as described previously
(Flaskos et al., 2007; Hargreaves et al., 2006; Harris et al., 2009a,
2009b; Sidiropoulou et al., 2009b). Cell viability was assessed by
monitoring the reduction of thiazolyl blue tetrazolium bromide (MTT)
using the method of Denizot and Lang (1986). Sublethal effects were
confirmed by the inability of CPO treatment to cause a decrease in MTT
reduction compared to the control.
Measurement of acetylcholinesterase activity
For this, cells were induced to differentiate as above, except that 2
million cells were plated into T75 culture flasks in 40 ml of culture
medium. Cells were harvested by centrifugation, resuspended in 20 ml
of PBS at 4 °C and recentrifuged to remove traces of DMEM. Cell pellets
were resuspended in 0.6 ml of 200 mM sodium phosphate buffer
(pH 7.4) at 4 °C and sonicated for 15 sec on ice. The activity of AChE in
cell sonicates was monitored by the method of Ellman et al. (1961)
modified for a microtitre plate format as described previously (Flaskos
et al., 2007; Sidiropoulou et al., 2009b). Protein was determined in the
sonicated extracts by the bicinchoninic acid (BCA) assay (Brown et al.,
332 J. Flaskos et al. / Toxicology and Applied Pharmacology 256 (2011) 330–336
1989) and specific activity was expressed as absorbance change /mg
protein/h.
Gel electrophoresis and Western blotting
For Western blotting analysis, cells were seeded and induced to
differentiate in the presence and absence of CPO in T75 culture flasks,
as described above. Intact cell monolayers were then solubilised by
boiling in 2 ml of 0.5% SDS in TBS prior to protein determination using
the BCA assay. The resultant cell lysates were subsequently subjected
to gel electrophoresis in the presence of sodium dodecyl sulphate
(SDS-PAGE) employing a 10% w/v polyacrylamide resolving gel,
overlaid with a 4% w/v polyacrylamide stacking gel (Laemmli, 1970).
The separated proteins were then electrophoretically transferred onto
nitrocellulose membrane filters (Towbin et al., 1979).
The resultant Western blots were blocked with 3% w/v BSA in TBS
(BSA/TBS) for at least 1 h at room temperature. Blots were then
probed overnight at 4 °C with appropriate dilutions of primary
antibodies in BSA/TBS, including anti-total NFH (N52; 1/500), anti-
pNFH (SMI34; 1/500), anti-GAP-43 (GAP7B10; 1/500), anti-α-tubulin
(B512; 1/2000). After six 10-min washes in TBS containing 0.05% v/v
Tween-20 (TBS/Tween), blots were probed with HRP-conjugated
anti-mouse immunoglobulin (1/2000) for 3 h at room temperature.
Following 6 further washes with TBS/Tween, antibody reactivity was
visualised with enhanced chemiluminescence reagent (ECL). For
quantification of antibody reactivity, band intensities on images of
developed Western blots were determined using Advanced Image
Data Analysis software (Fuji). Anti-tubulin reactivity levels were used
for normalisation.
Indirect immunofluorescence staining of cell monolayers
The intracellular distributions of tubulin, GAP-43 and NFH were
further studied by indirect immunofluorescence staining. Typically,
300 μl of N2a cell suspension (50,000 cells/ml) were seeded into each
well of an 8-well cell culture chamber slide. After 24 h differentiation
in the presence or absence of CPO, cells were fixed for 10 min at
−20 °C in 90% v/v methanol in TBS. They were then blocked with
BSA/TBS for 45 min at room temperature, extracted in TBS/Tween for
5 min followed by incubation with TBS for a further 5 min at room
temperature. Cells were then incubated overnight at 4 °C with
primary antibodies against tubulin (B512; 1/100), GAP-43
(GAP7B10; 1/50) or NFH (N52 and SMI34; both diluted 1/50 in
BSA/TBS). After three 5-min washes with TBS, they were incubated for
2 h at room temperature with FITC-conjugated rabbit anti-mouse IgG
secondary antibodies. After 3 further washes in TBS, cells were
mounted under glass cover slips using VectaShield® mounting
medium for fluorescence (Vector Laboratories Ltd., Peterborough,
UK) and viewed using a Leica CLSM confocal laser scanning
microscope fitted with epifluorescence optics, as described previously
(Flaskos et al., 2007; Hargreaves et al., 2006; Harris et al., 2009b).
Statistical analysis of data
All sets of data are based on a minimum of 3 independent
experiments and expressed as mean ± SEM. Average values for each
treatment were compared to the corresponding control by one-way
or two-way ANOVA, as appropriate, using 95% confidence limits.
Results
In order to obtain data comparable to our previous work on
organophosphorothionate toxicity, N2a cells were induced to differen-
tiate for up to 24 h in the presence of 0–10 μM CPO. Measurement of
MTT reduction indicated no significant decrease in CPO treated cells
compared to the controls (not shown). However, there was a
concentration-dependent reduction in the outgrowth of axon-like
neurites following 24 h exposure to the same levels of CPO (Fig. 2).
The acute cholinergic effects of CPO were investigated by performing
assays of AChE activity following exposure of differentiating N2a cells to
the same concentrations of CPO for 4 h and 24 h. As indicated in Fig. 3,
there was a statistically significant dose-dependent reduction in the
specific activity of AChE following both 4 h and 24 h exposure, with
values falling to approximately 30% of control values at 5 and 10 μM
(p b 0.02). After 24 h exposure, there appeared to be slightly less
inhibition at the lower concentrations of CPO than at 4 h, although this
effect was not deemed to be statistically significant when tested by two-
way ANOVA.
The molecular basis of the observed ability of CPO to inhibit neurite
outgrowth at sub-lethal concentrations was further investigated by
Western blotting analysis using antibodies to α-tubulin, GAP-43, NFH
and pNFH. As shown in the panel of blots in Fig. 4, the cross-reactivity
of cell lysates with monoclonal anti-α-tubulin antibody B512 was
apparently unaffected by exposure to CPO. Anti-GAP-43 (GAP7B10)
cross-reactivity appeared to be reduced compared to the control
following exposure to higher concentrations of CPO. Anti-NFH (N52)
also showed a trend of reduced cross-reactivity at increasing
concentrations of CPO, whereas reactivity of blots with anti-pNFH
(SMI34) showed slightly raised or similar levels to the control in
lysates of CPO treated cells.
Quantification of the observed changes was then performed using
AIDA software on blots obtained from 4 independent experiments. As in
previous work on OP treatment of N2a cells, anti-tubulin reactivity
showed no significant change following treatment with CPO (104% of
the control values at 10 μM CPO). Since tubulin was used for normal-
isation in previous studies on N2a cells, the densitometric values of all
other bands were normalised to tubulin, which served as a loading
control. The observed reductions in reactivity of cell lysates with anti-
GAP-43 and anti-NFH were confirmed by densitometric analysis, which
indicated a dose-dependent decrease in peak area in both cases that was
statistically significant compared to the non-CPO-treated control at the
highest concentration tested (Table 1).
In contrast to the decrease in reactivity with anti-NFH, cross-
reactivity with anti-pNFH was not significantly different from control
values (p N 0.8). In order to determine whether the different responses
observed for anti-NFH and anti-pNFH reflected altered phosphoryla-
tion status of NFH, band intensities relative to the control were
calculated as a ratio, where a value of 1 would indicate no overall
change in phosphorylation status. As indicated in the right hand
column of Table 1, there was a consistent dose-dependent decrease in
Fig. 2. Effects of chlorpyrifos oxon on neurite outgrowth in differentiating N2a cells.
Mouse N2a neuroblastoma cells were induced to differentiate in the absence and
presence of CPO, fixed, stained and assessed for the outgrowth of axon-like neurites as
described in Materials and methods. Shown are the mean numbers of axon-like
neurites per 100 cells ± SEM from 4 independent experiments. Asterisks indicate
statistically significant differences compared to the untreated control (p b 0.001).
 J. Flaskos et al. / Toxicology and Applied Pharmacology 256 (2011) 330–336
Fig. 3. Effects of chlorpyrifos oxon on acetylcholinesterase activity in differentiating N2a
cells. Mouse N2a neuroblastoma cells were induced to differentiate for 4 h or 24 h in the
absence and presence of CPO, harvested and cell sonicates assayed for AChE activity as
described in Materials and methods. Results are expressed as mean specific activity
(Absorbance change/mg protein/h) ± SEM for 3 (24 h) or 4 (4 h) independent
experiments. Asterisks indicate significant differences compared to the untreated
control (p b 0.02).
the phosphorylation ratio (NFH: pNFH), which was statistically
significant following exposure to both 5 μM and 10 μM CPO.
The effects of treatment with CPO on the intracellular distribution
of tubulin, NFH and GAP-43 were then studied by indirect immuno-
fluorescence staining of cells following 24 h exposure. The staining
Fig. 4. Detection of cytoskeletal and growth associated proteins on Western blots of N2a
cell lysates. Mouse N2a neuroblastoma cells were induced to differentiate for 24 h in
the absence and presence of CPO and cell lysates subjected to SDS-PAGE and Western
blotting as described in Materials and methods. Shown are typical blots probed with
antibodies to NFH (N52), pNFH (SMI34), GAP-43 (GAP7B10) and α-tubulin (B512),
followed by HRP-conjugated secondary antibodies and development using ECL reagent.
333
Table 1
Densitometric analysis of probed western blots. Lysates of N2a cells induced to
differentiate in the absence and presence of CPO were subjected to SDS-PAGE and
Western blotting analysis as described in Materials and methods. Shown are the
average band intensities ± SEM from a minimum of 4 independent experiments,
showing cross-reactivity with antibodies to GAP-43 (GAP7B10), total NFH (N52) and
pNFH (SMI34). The phosphorylation ratio of the change in NFH:pNFH is shown in the
right hand column of the table. Asterisks indicate changes that are statistically
significant compared to the non-CPO-treated control (p b 0.05).
CPO concentration Antibody reactivity (% control ± SEM)
(μM)
GAP43 NFH pNFH NFH:pNFH ratio
1 115.2 ± 35.4 84.5 ± 20.6 137.5 ± 34.4 0.64 ± 0.12
5 74.2 ± 17.5 58.8 ± 15.1 102.0 ± 22.4 0.59 ± 0.11*
10 23.6 ± 7.5* 42.3 ± 11.5* 119.0 ± 45.3 0.32 ± 0.06*
patterns for all antibodies in Fig. 5 showed a gradual decrease in
neurite outgrowth as the concentration of CPO increased. Staining
with anti-α-tubulin antibody was strong in both cell bodies and
neurites (when present) even at the highest concentration of CPO
(Figs. 5a–c). In the case of anti-GAP-43 staining, a general reduction in
staining intensity occurred at increasing CPO concentration with
weaker staining of neurites when present in CPO-treated cell
monolayers (Figs. 5d and e). Anti-NFH antibody N52 stained long
axon-like neurites strongly in control cells but much more weakly in
CPO-treated cells (Figs. 5g–i). The most predominant feature of CPO
treated cells stained with this antibody was the presence of
aggregates of anti-NFH staining (upper arrows in Figs. 5h and i).
The axonal staining pattern for ant-pNFH was similar to that of anti-
NFH in control cells; however, the cell body was much more evenly
stained with this antibody than with anti-NFH (Figs. 5j–l). The
staining intensity of anti-pNFH and its diffuse distribution in cell
bodies remained constant for all treatments, although as previously
mentioned the number of stained neurites decreased.
Discussion
In previous work we found that sub-lethal concentrations of
organophosphorothionate and organophosphonothionate pesticides
such as CPF, DZ and leptophos, inhibited the outgrowth of axon-like
processes by differentiating N2a cells (Flaskos et al., 2007; Sachana et
al., 2001, 2003). More recently, DZO was also found to inhibit neurite
outgrowth over the same concentration range. The aim of this study
was to determine whether CPO behaved in a comparable fashion and
to determine its effects on a similar panel of protein biomarkers to
that used in studies of the other compounds.
The inability of CPO to cause a decrease in MTT reduction by
differentiating N2a monolayers suggests that 1–10 μM CPO has no
significant effect on cell viability under the conditions tested. This
finding is in good agreement with our previous work, in which similar
concentrations of CPF, DZ and DZO were also found to be sub-lethal
towards differentiating N2a cells after 24 h exposure (Flaskos et al.,
2007; Sachana et al., 2001, 2005; Sidiropoulou et al., 2009b). However,
morphological analysis of Coomassie blue stained cells clearly in-
dicates that these concentrations of CPO caused inhibition of neurite
outgrowth in a dose dependent manner, which is again consistent with
our previous findings for CPF, DZ, leptophos and DZO (Flaskos et al.,
2007; Sachana et al., 2001, 2003, 2005; Sidiropoulou et al., 2009b).
The data obtained in this study showed significant inhibition of the
enzymatic activity of AChE both concomitantly and prior to the
morphological and biochemical effects of CPO. In a previous study with
N2a cells and the oxon metabolite of DZ, a significant AChE inhibition
was also noted, but only at the earlier time point (Sidiropoulou et al.,
2009b). The demonstration of a significant reduction in AChE activity
by CPO in this study both at 4 and 24 h is in line with previous data
showing that administration of CPF, compared to DZ under the same
334 J. Flaskos et al. / Toxicology and Applied Pharmacology 256 (2011) 330–336

Fig. 5. Indirect immunofluorescence staining of differentiating N2a cells. Mouse N2a neuroblastoma cells induced to differentiate for 24 h in the absence and presence of CPO, were
fixed and stained by indirect immunofluorescence as described in Materials and methods. Shown are typical images of control cells (a, d, g, k) and cells treated with 5 μM (b, e, h, l) or
10 μM (c, f, i, m) CPO stained with anti-α-tubulin (a–c), anti-GAP43 (d–f), anti-NFH (g–i) and anti-pNFH (k–m). Horizontal arrows indicate typical axon-like neurites in controls and
vertical arrows indicate typical aggregates detected by anti-NFH staining in CPO treated cells. Bar represents 20 μm.

conditions, produces greater inhibition of AChE (Jameson et al., 2007)
and higher mortality (Slotkin et al., 2006). However, on the basis of our
previous data on differentiating N2a cells, it is unlikely that the
inhibition of axon-like outgrowth is aetiologically related to AChE
inhibition. For example, although the OP trio-ortho-cresyl phosphate
is a weak inhibitor of AChE (Lock and Johnson, 1990), it inhibits
outgrowth of axon-like processes in N2a cells to an equal or even
greater extent than the AChE-inhibiting OPs, DZ and CPF (Flaskos et al.,
2007; Fowler et al., 2001; Sachana et al., 2005). That the inhibition of
the enzymatic activity of AChE by CPF is not related to its neurite
inhibitory effect has also been demonstrated in a number of cell
culture studies (Das and Barone, 1999; Howard et al., 2005) as well as
in vivo (Slotkin et al., 2006).
Previous studies showed that exposure to CPF, DZ and leptophos
was associated with reduced levels of NFH compared to the control
(Flaskos et al., 2007; Sachana et al., 2001, 2003, 2005). Densitometric
analysis of Western blots in the current work suggests that this is also
the case for CPO exposure, in contrast to DZO which was previously
shown to have no overall effect on total NFH levels (Sidiropoulou et
al., 2009b). Reduced levels of NFH detected on Western blots could
reflect increased degradation by proteases such as cathepsin D or
calpain (James et al., 1998; Nixon and Marotta, 1984). In this respect,
activation of calpain has been shown to occur in response to OP
treatment in vivo (El-Fawal and Ehrich, 1993). However, the reduced
antibody reactivity could also be caused by decreased synthesis of
NFH, which could be related to the known ability of CPF to inhibit the
synthesis of both DNA and proteins in vivo (Song et al., 1998).
Neurofilaments are known to be important in the regulation of
axon growth and stability and NFH becomes more highly expressed
and increasingly phosphorylated as axons mature (Lee et al., 1988; Lee
 J. Flaskos et al. / Toxicology and Applied Pharmacology 256 (2011) 330–336
and Cleveland, 1994; Veeranna et al., 1998). Previous work indicated
that DZO exposure was associated with increased phosphorylation of
NFH. However, the lack of a significant change in reactivity of lysates
with SMI34 suggests that, in contrast to the effects of DZO, there is no
overall change in the absolute levels of pNFH in CPO treated cells.
Although a significant increase was not observed in CPO treated cells in
the current work, the lack of change in SMI34 antibody reactivity
relative to the control is consistent with a possible alteration to the
overall phosphorylation status of NFH in CPO-treated cells. This notion
was confirmed by calculating the ratio of CPO induced change in NFH
to that in pNFH, which clearly shows a significant reduction to 59% and
32% of normal levels with 5 and 10 μM CPO, respectively, further
confirming the reduction in total NFH levels relative to pNFH. These
findings are consistent with the possibility that NFH is more prone to
proteolytic degradation when it is not phosphorylated and/or that the
non-degraded NFH in CPO-treated cells has an increased level of
phosphorylation compared to the control. Further work will help to
determine whether CPO is able to activate a protease or a protein
kinase for which NFH is a substrate and/or inhibit the activity of a
protein phosphatase that regulates the phosphorylation status of NFH.
Data obtained from the quantitative analysis of blots probed with
monoclonal antibody GAP7B10 suggested that there was a dose
dependent reduction in the levels of GAP-43 protein in lysates of CPO
treated cells. This finding is in good agreement with our previous work
in which N2a cells were exposed to a number of different OPs (Fowler et
al., 2001; Harris et al., 2009a; Sachana et al., 2001, 2003, 2005;
Sidiropoulou et al., 2009b), suggesting that GAP-43 represents a
common molecular marker of sub-lethal neurite inhibitory effects of
OPs. These changes in GAP-43 could have a significant bearing on the
morphological effects of CPO and other OPs, since GAP-43 is known to
exhibit increased synthesis and play a key role during axon outgrowth
(Meiri et al., 1986, 1998; Skene, 1989). Consistent with the aforemen-
tioned studies on N2a cells, tubulin levels were unaffected by any of the
CPO concentrations in the present study, which is in agreement with
findings in the above mentioned studies on N2a cells, suggesting that
microtubules are not a major target for these compounds in differen-
tiating N2a cells.
The CPO induced reduction in the number of neurites stained by
indirect immunofluorescence with antibodies to cytoskeletal proteins
confirmed the dose-dependent reduction shown in the morphological
analysis presented in Fig. 2. The staining patterns for monoclonal
antibodies B512, GAP7B10 and SMI34 suggested that the distribution
of tubulin, GAP-43 and pNFH, respectively, within the cell body of N2a
cells was not significantly affected by CPO treatment. The fact that,
even following exposure to 10 μM CPO, axon like neurites (when
present) were still strongly stained with the anti-tubulin antibody
B512 further supports the view that tubulin distribution and the
microtubule network are not significantly affected in this cellular
model. However, Grigoryan and Lockridge (2009) demonstrated that
CPO could bind to tubulin and disrupt the polymerisation of purified
bovine brain microtubules in vitro, suggesting that a direct interaction
of this compound with tubulin was possible. This apparent discrep-
ancy may be due to differences in the experimental procedures used;
thus, under the conditions tested, microtubules in differentiating N2a
cells may be less accessible to CPO binding than the purified bovine
brain microtubules used in the study of Grigoryan and Lockridge
(2009). Further work on the identification of CPO binding targets in
N2a cells would be worthwhile.
The observed decrease in relative staining intensity in axons for
GAP-43 and NFH further confirms that the levels of GAP-43 and NFH
in axons may be reduced in CPO-treated cells compared to the control.
Disruption of the neurofilament network is further confirmed by
major changes in the staining pattern of anti-NFH, which indicates a
major reorganisation of total NFH from axon staining in control cells
to cell body localised aggregates in CPO-treated cells. This type of
disruption is consistent with previous studies in our laboratory, in
335
which other OPs also caused reduced levels of total NFH and the
formation of aggregates in cell bodies (Flaskos et al., 1998, 2007;
Hargreaves et al., 2006), indicating that the two phenomena may be
closely related.
The above morphological and biochemical effects of CPO noted in
vitro are caused by concentrations similar to those occurring in vivo in
the developing organism. In humans, foetal concentrations of the
parent phosphorothionate CPF may commonly reach levels of 8 μg/ml
(22.8 μM) (Ostrea et al., 2002). As shown recently, the steady state
ratio of CPO/CPF in human plasma varies widely among adults and in
several cases can be higher than 0.04–0.05 (Eyer et al., 2009). Due to
the defective hydrolysis of the oxons by A- and B-esterases during
mammalian development (Furlong et al., 2006; Vidair, 2004), this
ratio is expected to be higher in the developing organism. Thus, levels
of CPO in the low micromolar range are attainable in developing
humans.
Although CPO is less lipophilic than CPF, which might affect its
penetration into the foetus, data showing considerable inhibition of
cholinesterase in foetal tissues after in vivo administration of
phosphorothionate insecticides (Gupta, 1995) indicate that the foetus
is exposed to the oxon. In humans, the foetus is exposed to CPO
generated in maternal tissues. In contrast, placental metabolism is
unlikely to contribute significantly to CPO formation, since CYP2B6,
the main enzyme responsible for converting CPF to CPO in humans
(Croom et al., 2010), is expressed to a very low extent in human
placenta (Pelkonen et al., 2006).
In conclusion, the data presented here suggest that exposure of
differentiating N2a cells to sub-lethal neurite inhibitory levels of CPO
is associated with persistent inhibition of AChE, reduced levels of
GAP-43 and NFH protein but that the level of NFH phosphorylation is
unaffected.
Conflict of interest statement
The authors do not have any conflicts of interest to disclose.
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