Wayne Harris

Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: identification of novel biomarkers of effect

In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells

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

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.

Diazinon oxon interferes with differentiation of rat C6 glioma cells.

The purpose of this study was to evaluate the toxicity of diazinon oxon (DZO), a major in vivo metabolite of the organophosphate insecticide diazinon (DZ), on differentiating rat C6 glioma cells. At concentrations shown to be non-cytotoxic by both the MTT and the Kenacid blue dye binding assays (1, 5 and 10 microM), DZO caused after 24h a reduction in the number of extensions developed from C6 cells induced to differentiate by serum withdrawal and addition of sodium butyrate. Densitometric scanning of Western blots of extracts of C6 cells demonstrated that, at all concentrations used, DZO decreased after 24h the expression of glial fibrillary acidic protein (GFAP) compared to controls. In addition, exposure to 10 microM DZO for 24h reduced the levels of tubulin and microtubule associated protein 1B (MAP1B). On the other hand, levels of MAP2c were not affected by DZO treatment. In contrast to our previous data on DZ, the above findings suggest that its oxon metabolite, DZO, may, at biologically relevant, subcytotoxic concentrations, interfere with glial cell differentiation.

Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines

The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca(2+)-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin. The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40-60% of control levels at sub-lethal concentrations (0.1 microM) that were approximately 100-fold lower than their IC(50) values in cytotoxicity assays. Following 24h exposure of N2a cells to 0.3 and 3 microM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 microM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl2 for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12 μM Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.

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.

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.

Diazoxon Disrupts the Expression and Distribution of βIII-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 β‐tubulin levels. However, probing with a monoclonal antibody that recognised specifically the βIII‐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 βIII‐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.

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 β-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.