Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2.

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.

The neuronal cytoskeleton as a potential target in the developmental neurotoxicity of organophosphorothionate insecticides.

Phosphorothionates are toxicologically the most important class of organophosphorus ester (OP) insecticides. Phosphorothionates are metabolically converted in vivo to their oxon analogues. These oxon metabolites can bind and inhibit acetylcholinesterase, thus causing acute cholinergic neurotoxicity. Oxon binding to the same target may also be partly responsible for manifestation of the 'intermediate syndrome'. More recent evidence suggests that the oxons may be also capable of inducing developmental neurotoxicity. The neuronal cytoskeleton may represent a potential target for the developmental neurotoxicity of the oxons because of its vital importance in many stages of normal neurodevelopment. Data obtained in the last five years and critically reviewed here indicate that the oxon metabolites, at concentrations that can be attained in vivo, exert potent effects on the neuronal cytoskeleton disrupting all three cytoskeletal networks. This disruption is expressed at the level of cytoskeletal protein expression, intracellular distribution, post-translational modification, cytoskeletal dynamics and function and may involve effects on both neuronal and glial cells. These effects are not secondary to other changes but may constitute primary effects of the oxons, as these compounds have been shown to be capable of covalently binding to and organophosphorylating multiple sites on tubulin and actin. Analogous studies must be extended to include other neurodevelopmentally important cytoskeletal proteins, such as neurofilament heavy chain, and tau, which are known to contain unusually high numbers of phosphorylatable sites and to establish whether organophosphorylation by the oxons takes place at sites where neurodevelopmentally relevant, endogenous, reversible phosphorylation is known to occur.

Diazoxon disrupts the expression and distribution of ßIII-tubulin and MAP 1B in differentiating N2a cells.

This study aimed at assessing the effects of diazoxon (DZO), a major metabolite of the insecticide diazinon (DZ), on key cytoskeletal proteins in differentiating N2a neuroblastoma cells. Initial experiments established that sublethal concentrations of 1, 5 and 10 µM DZO produced profound inhibition of neurite outgrowth. Densitometric scanning of probed immunoblots of N2a cell lysates demonstrated that DZO had no effect on total ß-tubulin levels. However, probing with a monoclonal antibody that recognised specifically the ßIII-tubulin isotype revealed that 10 µM DZO induced a significant reduction in the levels of this particular form. Levels of polyglutamylated tubulin were not altered. Exposure to 10 µM DZO also decreased the expression of microtubule-associated protein 1B (MAP 1B). However, DZO had no effect on the expression of MAP tau. DZO also failed to affect the levels neurofilament light (NFL) and neurofilament medium (NFM) chain levels. Indirect immunofluorescence demonstrated that the staining of neurites in treated cells was weaker than in the controls for ßIII-tubulin. In conclusion, DZO disrupts the microtubule (MT) network affecting the expression and distribution of two specific MT proteins known to be important in neuritogenesis. DZO may contribute to the developmental neurotoxicity seen following exposure to DZ.

The developmental neurotoxicity of organophosphorus insecticides: a direct role for the oxon metabolites.

Several extensively used organophosphorus ester (OP) insecticides are phosphorothionates. The oxon metabolites of phosphorothionates have long been known to be responsible for the acute cholinergic neurotoxicity associated with OP poisoning. In addition, there is now sufficient evidence to suggest that the oxon metabolites may also be directly responsible for the particular neurotoxicity that phosphorothionate insecticides, and especially chlorpyrifos (CP) and diazinon (DZ), are known to inflict on the developing organism. In vitro data reveal that the oxons, which are present at increased levels in the developing brain, have the ability to directly disrupt, at toxicologically relevant doses, separately a number of neurodevelopmental processes, including those of neuronal proliferation, neuronal differentiation, gliogenesis and apoptosis. In most cases, the effects of the oxons are very potent. Inhibition of neuronal and glial cell differentiation by the oxons in particular is up to 1000-times stronger than that caused by their parent phosphorothionates. The neurodevelopmental toxicity of the oxons is not related to the inhibition of the enzymatic activity of acetylcholinesterase (AChE), but may be due to direct oxon interference with the morphogenic activity that AChE normally shows during neurodevelopment. Other possible direct targets of the oxons include neurodevelopmentally important cell signaling molecules and cytoskeletal proteins which have been found to be affected by the oxons and to which covalent binding of the oxons has been recently shown. Future studies should aim at confirming the developmental neurotoxic capacity of the oxons under in vivo conditions and they must also be extended to include OP parent insecticides with a P=O moiety.

Ursodeoxycholic acid promotes intestinal adaptation in a cat model of short bowel syndrome.

The aim of this study was to assess the effect of ursodeoxycholic acid (UDCA) on the morphological and functional adaptive response of the jejunal remnant after massive intestinal resection in a cat model of short bowel syndrome (SBS). UDCA was administered to animals at a daily oral dose of 15 mg/kg for 6 weeks following a 85% jejunoileal resection. Resection alone caused extensive hyperplasia of jejunal mucosa, as evidenced by a significant increase in the weight of jejunal mucosa per unit length as well as by significant increases in DNA and protein concentration but no change in the protein/DNA ratio. Morphometric analysis using microscopy revealed no changes in jejunal mucosa thickness, jejunal crypt depth, villus height and villus surface area, although villus thickness was increased. The specific activities of jejunal sucrase and alkaline phosphatase were unaffected. UDCA treatment of resected animals, using doses that caused no toxicity, as evidenced by the absence of serum biochemistry abnormalities and histopathology, did not induce, compared to resection alone, any changes in mucosal cellularity and did not affect villus morphometry. On the other hand, UDCA administration increased crypt depth and, also, induced a profound increase in the specific activity of sucrase. UDCA improved diarrhoea, a core SBS symptom, reflected in a considerably reduced frequency of defaecation and improved form and texture of faeces. It is concluded that UDCA administration may enhance the natural adaptive response of the intestinal remnant following massive jejunoileal resection and may, thus, be beneficial in SBS treatment.

Fipronil interferes with the differentiation of mouse N2a neuroblastoma cells.

The purpose of this study was to evaluate the neurotoxic potential of the pesticide fipronil (FIP) towards the differentiation of mouse N2a neuroblastoma cells. At concentrations of 1, 5 and 10 µM that were not cytotoxic, as shown by two different cell viability assays, FIP impaired potently after 24h the development of axon-like processes, with a concentration of 1 µM causing 50% inhibition. Densitometric analysis of immunoblots of extracts of N2a cells exposed to FIP demonstrated that the axon-inhibitory action of the pesticide was not accompanied by significant changes in the levels of total and phosphorylated neurofilament heavy chain (NFH). FIP also induced no alteration in the levels of total and tyrosinated α-tubulin. On the other hand, this pesticide caused severe disruption of the developmentally important ERK 1/2-MAP kinase signal transduction pathway, as evidenced by significant reductions in the activation state of MAPK kinase (MEK 1/2) and, particularly, ERK 1/2. The above data seem to justify very recent concerns that FIP has the capacity to induce developmental neurotoxicity in mammals.

Neuroprotection from diazinon-induced toxicity in differentiating murine N2a neuroblastoma cells.

In previous work, the outgrowth of axon-like processes by differentiating mouse N2a neuroblastoma cells was shown to be inhibited by exposure to 10 microM diazinon. In the present work, N2a cells were induced to differentiate for 24 h in the presence and absence of 10 microM diazinon and 20% (v/v) conditioned medium derived from differentiating rat C6 glioma cells. Cells were then stained or lysed for morphological and biochemical analyses, respectively. The data showed that co-treatment with conditioned medium prevented the neurite inhibitory effect of diazinon. Furthermore, a significant recovery was also observed in the reduced levels of neurofilament heavy chain (NFH), heat shock protein-70 (HSP-70) and growth-associated protein-43 (GAP-43) observed as a result of diazinon treatment in the absence of conditioned medium, as seen by densitometric analysis of Western blots of cell lysates probed with monoclonal antibodies N52, BRM-22 and GAP-7B10. By contrast, no significant change was noted in the reactivity of cell lysates with antibodies against alpha- and beta-tubulin under any condition tested. After pre-incubation with a polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) antibody, conditioned medium derived from rat C6 glioma cells lost its ability to protect N2a cells against the neurite inhibitory effects of diazinon. In conclusion, these data demonstrate that C6 conditioned medium protects N2a cells from the neurite inhibitory effects of diazinon by blocking molecular events leading to axon damage and that GDNF is implicated in these effects.

Diazinon oxon affects the differentiation of mouse N2a neuroblastoma cells.

The aim of this study was to assess the neurotoxicity of diazinon oxon (DZO), a major in vivo metabolite of the phosphorothionate insecticide diazinon (DZ), on differentiating mouse N2a neuroblastoma cells. When used at concentrations of 1, 5 and 10 microM, DZO did not cause cell death but it impaired the outgrowth of axon-like processes after 24 h. Densitometric scanning of Western blots of lysates of N2a cells revealed that exposure to 5 or 10 microM DZO for 24 h increased the expression of phosphorylated neurofilament heavy chain (NFH) compared to controls, while there was no significant change in total NFH. By contrast, treatment of N2a cells with 1-10 microM DZO resulted in marked reductions in the expression of the axon growth-associated protein GAP-43. DZO-treated cells also showed an increased expression of the heat shock protein HSP-70 compared to controls. The above biochemical changes were not temporally related to inhibition of acetylcholinesterase (AChE). These data suggest that biologically relevant, subcytotoxic levels of DZO may exert neurotoxic effects on differentiating cells and that the mechanisms involved are different from those attributed to its parent compound.

Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity.

Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC(50) 2.5 microM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5 microM PSP for 4 h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24 h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-alpha-tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4 h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN).

Effects of phenyl saligenin phosphate on phosphorylation of pig brain tubulin in vitro.

Phenyl saligenin phosphate (PSP) induces a characteristic neuropathy (OPIDN), the molecular basis of which has not been precisely defined. This study examined the in vitro effects of PSP on the phosphorylation of serine and threonine residues of proteins in porcine brain cytosol. Quantitative analysis of Western blots probed with antibodies recognizing phosphorylated serine residues demonstrated that 100µM PSP induced a significant increase in the phosphorylation of serine residues of a 50kDa protein. This protein was identified as the α- and ß-tubulin subunits by probing Western blots of extracts separated by two-dimensional polyacrylamide gel electrophoresis with anti-phosphoserine and anti-tubulin antibodies. By contrast, threonine phosphorylation of the 50kDa polypeptide and other proteins detected on Western blots probed with anti-phosphothreonine antibodies, was not significantly affected by PSP. These data indicate that PSP is able to induce increased phosphorylation of tubulin in serine residues, consistent with a possible role for this phenomenon in OPIDN induction.

Effects of Chlorpyrifos and Chlorpyrifos-Methyl on the Outgrowth of Axon-Like Processes, Tubulin, and GAP-43 in N2a Cells.

The aim of this work was to study the neurodegenerative effects of the organophosphate (OP) pesticides chlorpyrifos (CPF) and chlorpyrifos-methyl (CHM) on cultured mouse N2a neuroblastoma cells. CPF or CHM, at a subcytotoxic concentration of 3 muM, were added to the cells either at the time of the induction of cell differentiation (codifferentiation) or 16 h after the induction of differentiation (postdifferentiation). CPF and CHM were similar in inhibiting significantly the outgrowth of axon-like processes from N2a cells after only 4 h exposure under both co- and postdifferentiation exposure conditions. Densitometric scanning of Western blots of extracts of cells treated with CPF or CHM for 4 h revealed significantly decreased cross-reactivity with a monoclonal antibody recognizing the protein GAP-43 under post- but not under codifferentiation exposure conditions. Exposure to CPF or CHM for 4 h under postdifferentiation conditions also resulted in reduced fluorescence of N2a cell body staining with anti-GAP-43. Cross-reactivity of Western blots with a monoclonal antibody recognizing alpha-tubulin was not significantly affected by OP treatment. These data indicate that a disturbance in GAP-43 may be important in the retraction of axons in predifferentiated N2a cells and support the notion that the mechanisms involved in CPF-and CHM-induced inhibition of axonal outgrowth may be different under co- and postdifferentiation exposure conditions.