Alan J. Hargreaves

The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells

The aim of this work was to study the effects of chlorpyrifos (CPF) on the outgrowth of axons by differentiating mouse N2a neuroblastoma cells. This was achieved by morphological, Western blotting and enzymatic analyses of cells induced to differentiate in the presence and absence of CPF added either at the same time (co-differentiation) or 16 h after (post-differentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 h exposure to CPF in both co- and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8 h of exposure but no significant effect at 4 h under both co- and post-differentiation conditions. By contrast, levels of the heat shock protein HSP-70 were raised at both time points, but only in co-differentiation experiments. Neuropathy target esterase (NTE) activity was lower than controls following 4 or 8 h of exposure under co-differentiation conditions, but not under any post-differentiation conditions. The results suggest that the inhibition of axon production and maintenance by CPF in differentiating N2a cells may involve multiple targets, which are different under co- and post-differentiation conditions.

Tricresyl phosphate inhibits the formation of axon-like processes and disrupts neurofilaments in cultured mouse N2a and rat PC12 cells

Tricresyl phosphate (1 microg/ml) inhibited the outgrowth of axon-like processes in mouse N2a neuroblastoma and rat PC12 pheochromocytoma cell lines induced to differentiate by serum withdrawal and nerve growth factor addition, respectively. By contrast, it had no effect on the outgrowth of processes by rat C6 glioma cells induced to differentiate with sodium butyrate. The effect on axon outgrowth in the two neuronal cell lines correlated with altered distribution of neurofilament proteins, as determined by indirect immunofluorescence with monoclonal antibody RMd09. Western blots of neuronal cell extracts probed with the same antibody revealed decreased cross-reactivity after exposure to tricresyl phosphate. The results suggest that tricresyl phosphate has a selective effect on neuronal cell differentiation, which involves impaired axon outgrowth, reduced levels of the neurofilament heavy chain and disruption of the neurofilament network.

The toxicity of organophosphate compounds towards cultured PC12 cells

The effects of three representative organophosphates (OPs), tricresyl phosphate (TCP), triphenyl phosphite (TPP) and paraoxon (POX) on the proliferation and viability of rat PC12 pheochromocytoma cells were studied. With respect to its IC50, TCP was at least an order of magnitude more potent in its antiproliferative activity than both TPP and POX. All test OPs were cytotoxic at concentrations inhibiting cell proliferation. No compound inhibited cell growth below 10 micrograms/ml. For TCP and TPP the estimated IC50 values from proliferation assays were lower than published LD50 values in vivo, whereas paraoxon was much less toxic in vitro than in vivo. Subcytotoxic levels of TCP (1 micrograms/ml) were found to inhibit the maintenance of neurites on cells grown in the presence of nerve growth factor.

Putative adverse outcome pathways relevant to neurotoxicity

Abstract The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two‐dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose‐6‐phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G‐actin to form F‐actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet‐derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments

Aims: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481.

Protection from MPTP‐induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF‐H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 µm) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP‐induced cell death (cytotoxicity) and MPTP‐induced NF‐H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 µm) reduced the cytotoxic effect of MPTP, but blocked di‐butyryl cyclic AMP (dbcAMP)‐induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine osidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP‐induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF‐H hyper‐phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP‐induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.

Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells

The aim of this work was to investigate the sublethal neuropathic effects of tricresyl phosphate (TCP: mixed isomers), triorthocresyl phosphate (TOCP) and triparacresyl phosphate (TPCP) on differentiating mouse N2a neuroblastoma cells. This was achieved by a combination of measurements of cell viability, axon outgrowth and the levels of cytoskeletal proteins detectable on western blots of extracts from cells induced to differentiate in the presence and absence of the compounds. In a time‐course experiment TCP inhibited the outgrowth of axon‐like processes following exposure times of 24 h or longer. Dose–response experiments indicated that TCP and TOCP exhibited similar sustained levels of toxicity following both 24 and 48 h exposure, with no significant difference between their respective IC50 values. By contrast, TPCP demonstrated a transient effect on the outgrowth of axon‐like processes, which was detectable after 24 but not 48 h of exposure. Isomer‐specific patterns of toxicity were also evident at earlier time‐points, with only the ortho isomer showing significant levels of inhibition of axon outgrowth following 4–8 h exposure. Probing of western blots with antibodies against cytoskeletal proteins indicated that the inhibition of axon outgrowth by these compounds was associated with a sustained reduction in the levels of phosphorylated neurofilament heavy chain. The inhibitory effect on axon outgrowth of TOCP but not TPCP was enhanced in the presence of a microsomal activation system. Since TOCP is the most neuropathic of the isomers of TCP in vivo, differentiating N2a cells provide a useful cellular system for mechanistic studies of the neurodegenerative effects of this organophosphate.

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Inhibition of neurite outgrowth in N2a cells by leptophos and carbaryl: effects on neurofilament heavy chain, GAP-43 and HSP-70.

The neurodegenerative properties of the organophosphate ester leptophos (LEP) and the carbamate ester carbaryl (CB), both of which can cause neuropathic effects in animals, were investigated in differentiating mouse N2a neuroblastoma cells. At a sublethal concentration of 3 microM, both LEP and CB were able to inhibit the outgrowth of axon-like processes from N2a cells after only 4 h of exposure. Extracts of cells exposed to LEP showed decreased cross-reactivities with monoclonal antibodies that recognise the neurofilament heavy chain (NFH) and the growth-associated protein GAP-43. However, they exhibited increased cross-reactivity with a monoclonal antibody that recognises the heat shock protein HSP-70. In contrast, no changes were noted in the levels of antibody binding in blots of extracts of cells exposed to CB. It is concluded that, although both LEP and CB inhibit the formation of axons in vitro, the early biochemical changes underlying the neurodegenerative effects of the two compounds are different.