Basic & Clinical Pharmacology & Toxicology, 2014, 115, 201–208

Doi: 10.1111/bcpt.12204

MiniReview

The Neuronal Cytoskeleton as a Potential Target in the Developmental Neurotoxicity of Organophosphorothionate Insecticides John Flaskos School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece (Received 14 November 2013; Accepted 14 January 2014) Abstract: 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.

Introduction – The Phosphorothionate Insecticides and the Neurotoxicological Implications of their Metabolism The organophosphorus esters (OPs) represent the largest group of insecticides globally employed. Although OPs are generally very effective against insects, they also show considerable toxicity to non-target animal species mainly affecting the nervous system. The adverse effects of OPs on this system encompass an impressive range of symptoms, and a number of distinct neurotoxicities have been delineated. These include an acute neurotoxicity characterized by symptoms typical of cholinergic hyperstimulation [1], organophosphate-induced delayed (poly) neuropathy [2,3], the ‘intermediate syndrome’ [4] and a characteristic persistent, long-term chronic neurotoxicity [5]. Of these neurotoxicities, the most frequent and fatal is the acute cholinergic neurotoxicity. Its molecular basis is the inhibition of the catalytic activity of acetylcholinesterase (AChE) in central and peripheral nerves. The inhibition of AChE may be also aetiologically important in the intermediate syndrome, as this generally occurs after prolonged and severe inhibition of Author for correspondence: John Flaskos, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece (e-mail [email protected]).

the enzyme, although not all patients showing such inhibition develop the syndrome [6,7]. According to the nature of the atoms directly attached to the central phosphorus atom, OP insecticides can be divided into 12 different classes [8]. Among these, the phospho(ro)thio (n)ate class includes compounds in which all three atoms that are directly attached to the phosphorus by single bonds are oxygen, whereas the atom to which the phosphorus is linked by a co-ordinate bond (commonly depicted as a double bond) is sulphur (Fig. 1). Phosphorothionates constitute the most important, from the toxicological point of view, class of OP insecticides. This class contains well over a dozen different insecticidal compounds including some that are among the most extensively used and/or involved in poisoning cases around the world, for example chlorpyrifos (CP), diazinon (DZ), methyl parathion, parathion, fenitrothion and others. Inspection of recently reviewed epidemiological data from a number of developed and less developed countries [9] indicates that a phosphorothionate consistently lies among the three OP insecticides mostly used and/or involved in poisoning. After exposure, the absorbed phosphorothionate is subjected to several different initial biotransformations. One of these involves the oxidative desulphuration of the phosphorothionate

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O

O Fig. 1. Chemical structure of phosphorothionates.

insecticide leading to the formation of its oxygen (oxon) analogue. This reaction is carried out exclusively by cytochrome P450-dependent monooxygenases which reside mainly in the liver. Phosphorothionate insecticide desulphuration leading to oxon formation is mainly mediated in human beings through the action of CYP3A4 and, particularly, CYP2B6 [10,11]. CYP2B6 is prone to polymorphism and interindividual differences in its expression are among the largest of the cytochromes P450, with CYP2B6 hepatic protein levels varying over 350 times [12]. This wide variability contributes to the large differences in oxon levels noted [13], potentially leading to different clinical outcomes (see below) and responses to antidotes after phosphorothionate insecticide intoxication. Although the oxidative desulphuration reaction simply involves replacement of the sulphur of the P=S group in the phosphorothionate molecule by oxygen, it has dramatic toxicological repercussions for the organism. This is basically due to the fact that the oxon analogue of the phosphorothionate formed is a very strong inhibitor of the catalytic activity of AChE, its inhibitory potency being up to 1000 times higher than that of the parent phosphorothionate. Thus, the oxon metabolites of the phosphorothionate insecticides are the compounds that are mainly responsible for the classical acute cholinergic neurotoxic effects commonly noted after phosphorothionate OP intoxication. As strong inhibition of AChE may be also important for the manifestation of the intermediate syndrome and may possibly account even for some of the neurobehavioural symptoms seen in chronic OP neurotoxicity, the range of neurotoxic effects that the oxon metabolites can precipitate in the organism can be considerably wide. More recently, yet another kind of neurotoxicity involving OP insecticides has been revealed. This neurotoxicity involves toxic effects specifically on the developing nervous system and has very severe socio-economic repercussions. The relevant evidence has been largely derived from experimental studies and concerns exclusively the phosphorothionate class and particularly CP and DZ. The demonstration of the potential of these phosphorothionates to cause developmental neurotoxicity in these experimental studies has been instrumental in the restriction of their use by regulatory bodies [14,15]. The developmental neurotoxicity of phosphorothionates comprises two distinct components. One of these involves the manifestation in the young of increased cholinergic toxicity as a result of greater AChE inhibition. This is mainly due to the higher concentrations of the oxon metabolites in the developing organism, as a result of a diminished capacity to convert the oxons to inactive metabolites by A- and B-esterases [16].

More recently, a more important component of the developmental neurotoxicity of phosphorothionates has emerged. This relates to the ability of phosphorothionates to disrupt phenomena and mechanisms normally taking place during neurodevelopment. Phosphorothionates can, in fact, interfere separately with each of the developmental processes of neuronal proliferation, differentiation, axonogenesis, synaptogenesis and apoptosis and can also disrupt both the proliferation and differentiation of glial cells [17]. Experimental data from a series of studies conducted by the research group of Slotkin have suggested that it is the phosphorothionate compound to which the organism is initially exposed that is directly responsible for the disrupting effects on neurodevelopment [18,19]. Nevertheless, a number of studies performed by separate research groups now indicate that the oxon metabolites of phosphorothionates have themselves the capacity to interfere directly with normal neurodevelopment and may, thus, induce developmental neurotoxicity. The relevant evidence has mainly derived from in vitro experiments employing cell cultures. For a detailed account of these investigations, the reader can be referred to a recent review [20]. In brief, the obtained data demonstrate that the oxon metabolites are capable of disrupting separately the developmental processes of neuronal cell replication [21], differentiation, neurite/axonal/dendritic outgrowth [22–26] and apoptosis [27]. In addition, the oxon metabolites are able to interfere with the processes of proliferation and differentiation in glial cells [21,28–30]. These effects are produced by oxon levels similar to those occurring in vivo in the developing organism [20] and, in some cases, by oxon concentrations as low as 0.001 nM [23,31]. The main part of this MiniReview that follows will focus on the targets of the developmental neurotoxicity of the oxons and specifically on the cytoskeleton. Targets of the Oxon Metabolites The oxon metabolites of phosphorothionate insecticides are highly reactive compounds. Thus, they show a much greater reactivity against such targets as AChE and many other (approximately 30) serine hydrolases [32] than their parent phosphorothionates. This is due to the fact that the oxygen of the oxons has greater electronegativity and electron-withdrawing capacity than the sulphur of the phosphorothionates. As a result, there is greater polarization of the P=O bonding compared with the P=S bonding. This renders the central phosphorus atom highly electrophilic and, thus, more susceptible to nucleophilic attack by the oxygen of the hydroxyl group of the active site serine of AChE and of the other serine hydrolases. Thus, the oxon metabolites have a much higher capacity to bind to and phosphorylate these enzymes. Additional targets of the oxons can be various lipases, for example diacylglycerol lipase [33], as these enzymes show a notable sequence homology with AChE near their active site serine [34]. Oxons can also interact and bind to cAMP [35] as well as to muscarinic [36,37], nicotinic [38] and cannabinoid CB1 [32] receptors. More recently, the oxon metabolites have been shown to be capable of covalently binding to proteins

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with no active site serines such as transferrin [39] and albumin [40]. However, all these studies have not been conducted in a neurodevelopmental context, and the relevance of the above oxon targets to the neurodevelopmental process is not clear. The Neuronal Cytoskeleton as a Target for the Developmental Neurotoxicity of the Oxon Metabolites: Theoretical Considerations The neuronal cytoskeleton is a good candidate for being a target for substances disrupting normal neurodevelopment. This stems from the fact that the cytoskeleton is essential for the various phases of the developmental process in the nervous system. Thus, any toxicity exerted towards the cytoskeleton might also inflict damaging effects on neurodevelopment. Developmentally important phenomena known to be critically dependent on the involvement of the cytoskeleton include cell proliferation/mitosis, cell motility/migration, establishment and maintenance of cell shape/morphology, cell differentiation, modulation of axonal radial growth, neurite outgrowth, extension of axons, initiation and elongation of axons, maintenance/ stabilization/inhibition of retraction of preformed axons, extension of dendrites, dendritic arborization, growth cone steering and advance and apoptosis. Apart from having good reasons for believing that the cytoskeleton may be a potential target for developmental neurotoxicants in general, there are also good reasons for reckoning that several important proteins of the neuronal cytoskeleton can be appropriate targets which the oxon metabolites of the phosphorothionates in particular can bind to and phosphorylate. Indeed, certain cytoskeletal proteins with a key role in a variety of crucial neurodevelopmental phenomena are known to contain in their molecules an unusually high number of phosphorylatable sites which can therefore be targeted and (organo) phosphorylated by the oxons. Thus, the main proteins of the neurofilament (NF) network, and particularly neurofilament medium chain (NFM) and neurofilament heavy chain (NFH), contain numerous serine residues, mainly in the carboxyl terminal multiple repeats of Lys-Ser-Pro (KSP) sequences, although other serine (or threonine) residues in the carboxyl terminal tail as well as in the amino terminal head domains of NFM and NFH can also be phosphorylated. Thus, NFM and NFH can carry 15–26 and 30–60 phosphate groups/ molecule, respectively [41], although the stoichiometry of in vivo phosphorylation varies widely depending on the physiological or pathological setting [42]. Particularly high numbers of phosphorylatable sites are also found in two important proteins of the microtubule (MT) network, MAP 2 and tau. These two proteins, which have a high carboxyl terminal homology, contain variants of the KSP repeat motif and their serine residues are substrates for phosphorylation. MAP 2 from brain prepared under certain conditions contains more than 30 phosphate groups per molecule [43], whereas human brain tau has 80 serine/threonine residues and five tyrosine residues and, thus, nearly 20% of the molecule represents potential sites for phosphorylation [44]. Although the physiological significance of most of the phosphorylatable sites that are present in the

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above cytoskeletal proteins is not clear, the phosphorylation and dephosphorylation of some defined sites by endogenous kinases and phosphatases are known to have a considerable bearing on the properties and functions of these proteins and some of these functions are relevant to neurodevelopmental phenomena, for example neurite outgrowth [42,45]. Thus, phosphorylation of these critical sites by the oxon metabolites of the phosphorothionate insecticides might be expected to lead to interference with normal neurodevelopment. The Effects of the Oxon Metabolites on the Neuronal Cytoskeleton Microtubules (MTs). Of the three components of neuronal cytoskeleton, MTs have received the greatest attention as a target for the oxons. Thus, in neonatal rat hippocampal slice cultures, 10 lM chlorpyrifos oxon (CPO) has no effect on a-tubulin levels, but induces significant, 12–18 per cent reductions in the levels of MAP 2, before cytotoxic effects are noted [46]. In the same rat preparation, CPO inhibits the polymerization of both purified and mitogen-activated protein (MAP)-enriched tubulin, with effects on the latter being two times stronger. CPO also interferes with the polymerization of bovine brain tubulin, as determined by atomic force microscopy nanoimaging [47]. Thus, biologically relevant concentrations of 5 and 10 lM CPO reduce the number as well as the length and the width of MTs, whereas higher CPO levels (25–100 lM) either stimulate polymerization or cause aggregation of MTs. In vivo administration of CPO in a single sublethal dose to mice also leads to a considerable decrease in the dimensions of brain MTs [48]. As nanoimaging shows, these MTs have fewer attached proteins. Mass spectroscopy has identified six missing proteins which include MAP 2, isoform 1. As this protein is particularly highly expressed during neurodevelopment and is important for axonal or dendritic outgrowth [49], the above data may have significant implications for the ability of CPO to induce developmental neurotoxicity. Another aspect of the ability of CPO to interfere with the function of the MT system is its capacity to interact with the MT-associated motor protein kinesin. Thus, in an in vitro MT motility assay, CPO, at low micromolar concentrations, induces an increase in the number of MTs detaching from kinesin, apparently weakening kinesin–MT interactions [51]. This CPO effect is 4.5 times stronger than that of its parent phosphorothionate, CP. These data imply that the normal kinesin-dependent intracellular movement of proteins, organelles and vesicles along MTs may be seriously compromised by CPO. Disruption of kinesin function would be expected to have especially severe repercussions during neurodevelopment as developing neurons are particularly dependent on the proper transport of necessary cargos from the cell body to the growing axons and synapses and also because of the known involvement of kinesin in the alignment/segregation of chromosomes during mitosis [51]. The above studies, summarized in table 1, indicate that levels of CPO that can be found in vivo may disrupt the

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structure, function and polymerization of MTs. However, with the exception of the study of [46], who used hippocampal slice cultures prepared from neonatal animals, these studies have not been carried out in a neurodevelopmental context. In contrast, a series of systematic investigations conducted by our research group (table 1) have examined effects on the MT system under neurodevelopmentally relevant conditions. In addition, in our studies, we have sought to assess such effects separately on neuronal and glial cells. Furthermore, to expand our knowledge on the behaviour of the oxon metabolites of phosphorothionate insecticides, we have not restricted ourselves exclusively to the study of CPO. In view of these, we have assessed the effects of CPO as well as those of the oxon metabolite of DZ, diazoxon (DZO), on the MT system adopting the use of both neuronotypic and gliotypic cell lines undergoing the process of differentiation. To this end, we have employed throughout our studies the mouse N2a neuroblastoma and the rat C6 glioma cell lines. These cell lines are commercially available, are easy to grow and differentiate and have proved useful in our study of the various effects of OPs, including those exerted on the neuronal and glial cytoskeleton [52]. In almost all cases, exposure of N2a and C6 cells to CPO and DZO has been for a period of 24 hr and the oxon concentrations employed have been in the range 1–10 lM. These concentrations can be attained in vivo and are subcyto-

toxic, as shown by such cytotoxicity assays as the MTT reduction assay [53] and the FRAME Kenacid blue bye binding assay [54]. Induction of differentiation of mitotic N2a cells has been attained by the withdrawal of serum from the growth medium and the subsequent addition of 0.3 mM dibutyryl cAMP, whereas mitotic C6 cells have been induced to differentiate by serum withdrawal and the addition of 2 mM sodium butyrate [52]. Our results on the impact of the oxon metabolites on the MT system in the neuronotypic N2a cell line show that CPO, at concentrations of 1–10 lM, has no significant effect on the levels of total a-tubulin [26]. In addition, CPO induces no changes in the distribution of a-tubulin within the N2a cells. Equimolar concentrations of DZO have also been found to exert no effect on total a-tubulin levels [25]. Interestingly, levels of a post-translationally modified, tyrosinated form of a-tubulin, which is important for the proper time control of neurite outgrowth and axonal differentiation [55], are also unaffected by DZO. More recent experiments show that DZO causes no change either in the levels of total b-tubulin [56]. On the other hand, under identical conditions, DZO, at a concentration of 10 lΜ, induces a significant 39% reduction specifically in the levels of the isotype III of b-tubulin. In addition, 10 lM DZO can affect the intracellular distribution of bIII-tubulin. This neuronal-specific isotype of b-tubulin

Table 1. Studies on the effects of the oxons on the cytoskeleton. Oxon Microtubules CPO CPO CPO CPO CPO CPO, DZO DZO DZO DZO DZO DZO

Concentration (lΜ) 10 3 mg/kg i.p. 10 5,10 1–10 1–10 1–10 1–10 10 10 1–10

CPO 1–10 DZO 10 CPO, DZO 10 CPO, DZO 1–10 Intermediate filaments CPO 10 DZO 5, 10 DZO 1–10 DZO 1–10 Microfilaments DZO 1–10

Preparation

Cytoskeletal effect

References

Neonatal rat hippocampal slices Mouse brain Neonatal rat hippocampal slices Bovine brain Bovine brain Differentiating mouse N2a cells Differentiating mouse N2a cells Differentiating mouse N2a cells Differentiating mouse N2a cells Differentiating mouse N2a cells Differentiating mouse N2a cells

↓MAP2, ↔ a-tubulin ↓MAP2, isoform 1 ↓tubulin polymerization ↓MT number and dimensions ↓kinesin – MT interaction ↔ a-tubulin ↔ tyrosinated a-tubulin ↔ b-tubulin ↓bIII- tubulin, bIII- tubulin distribution altered ↓MAP 1B, ↔ tau ↔ total and phospho stathmin

Differentiating Differentiating Differentiating Differentiating

rat rat rat rat

↓a-tubulin, a-tubulin distribution altered ↓a-tubulin ↓MAP1B ↔ MAP2c

[46] [48] [46] [47] [50] [25,26] [25] [56] [55] [55] [John Flaskos, Alan Hargreaves & Magda Sachana, Unpublished data] [29] [30] [29,30] [29,30]

Differentiating Differentiating Differentiating Differentiating

mouse N2a cells mouse N2a cells mouse N2a cells rat C6 cells

↓ total NFH, ↔ phospho NFH ↔ total NFH, ↑ phospho NFH ↔ total NFL, NFM ↓GFAP

[26] [25] [56] [30]

↔ total cofilin, ↑ phospho cofilin

[John Flaskos, Alan Hargreaves & Magda Sachana, Unpublished data]

C6 C6 C6 C6

cells cells cells cells

Differentiating mouse N2a cells

↑, increase; ↓, decrease; ↔, no change; CPO, chlorpyrifos oxon; DZO, diazoxon; GFAP, glial fibrillary acidic protein; MT, microtubule; NFH, neurofilament heavy chain; NFL, neurofilament light chain; NFM, neurofilament medium chain.

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represents one of the earliest cytoskeletal proteins to be expressed during development [57,58] and is critically involved in neuritogenesis [59]. Apart from its effect on the core MT protein tubulin, DZO can also affect certain MT-associated proteins. Thus, in parallel to its decreasing effect on bIII-tubulin levels, 10 lΜ DZO also induces in differentiating N2a cells a significant 49% reduction in the levels of MAP 1B [56]. Significantly, MAP 1B is normally the first MAP to be expressed in neurons [49] and is crucially involved in neuronal differentiation and the early stages of neuritogenesis [60]. On the other hand, in differentiating N2a cells, DZO exerts no effect on the levels of the MT-associated protein tau. Preliminary data from our laboratory show that DZO may also have no effect on either the levels or the phosphorylation status of the tubulin-binding protein stathmin, both of which are important for regulating MT dynamics that are necessary for neuronal development [61]. Data from our experiments with differentiating C6 cells indicate that CPO and DZO may disrupt during development the MT system also in glial cells. Thus, CPO levels of 1–10 lM induce in C6 cells a significant concentration-dependent decrease in the levels of a-tubulin, with 10 lΜ CPO causing a reduction of more than 75% [29]. As immunofluorescence staining reveals, 10 lΜ CPO can also disrupt potently the integrity of the MT network. Apart from CPO, DZO also impairs in C6 cells the expression of a-tubulin with a concentration of 10 lM inducing a significant 32% reduction [30]. These marked effects of oxon metabolites on a-tubulin in C6 cells are in sharp contrast to their lack of an effect in N2a cells (see previously). In addition to their considerable effect on a-tubulin, both oxons, at a level of 10 lΜ, also reduce in C6 cells the levels of MAP 1B [29,30]. On the other hand, the two oxons have no effect on the expression of the MT-associated protein MAP 2c, an immature form of MAP 2 found in differentiating neurons, glia and C6 cells [49,62]. Intermediate filaments (IFs) and microfilaments (MFs). In full appreciation of the fundamental role that the cytoskeleton plays in the neurodevelopmental process, we have not restricted our studies to the MT system, but have investigated the effects of the oxon metabolites also on the IF and the MF networks (table 1). Intermediate filament parameters assessed have included NF protein and glial fibrillary acidic protein (GFAP) levels, NF protein distribution and, also, NF protein phosphorylation, as the latter is known to be the main factor in the regulation of NF dynamics and function [42]. Thus, in differentiating N2a cells, CPO, at a concentration of 10 lM, has been found to reduce significantly the total levels of the NFH by 58% [26]. On the other hand, under these conditions, there is no change in the levels of phosphorylated NFH, implying that the overall phosphorylation status of NFH may be altered by CPO. In addition, CPO induces a major change in the distribution of total NFH within N2a cells leading to the formation of cell body aggregates. DZO also affects the NF network in differentiating N2a cells but in a different way. Thus, it induces no change in the total levels of NFH, while,

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at a concentration of 5 and 10 lΜ, causes significant 30–40% increases in the levels of phosphorylated NFH [25]. Total levels of NFM and light (NFL) chains are also unaffected by DZO [56]. The major IF protein in astroglia is also affected by DZO. Thus, in differentiating C6 cells, DZO, at a concentration of 1–10 lM, induces significant reductions in the expression of GFAP, an effect indicative of specific impairment of glial cell development in vivo [30]. We have recently embarked on exploring in the differentiating N2a cell line the effect of the oxon metabolites on the MF network. Preliminary data that have been obtained indicate that DZO, at concentrations of 1–10 lM, has no effect on the total levels of the actin-binding protein cofilin, which has an essential role as a regulator of actin filament dynamics during nervous system development [63]. On the other hand, these concentrations of DZO induced marked elevations specifically in the levels of the phosphorylated, inactive form of cofilin, with 1 lΜ DZO causing an almost 150% increase. Such changes should severely impair the ability of cofilin to promote growth cone motility and neurite extension [64], and they are in line with our data showing that 1–10 lΜ CPO concentrations cause drastic inhibition of neurite outgrowth in N2a cells [25]. Interestingly, a disruption in the normal balance between the phosphorylated and non-phosphorylated form of cofilin is noted with methylmercury, a well-established developmental neurotoxicant [65].

The Direct Targeting of Cytoskeletal Proteins by the Oxons The data presented above indicate that the oxon metabolites of phosphorothionate insecticides can have multiple disrupting effects on the neuronal cytoskeletal network during neurodevelopment. A critical mechanistic issue is whether the observed effects on the cytoskeletal proteins represent primary effects of the oxon metabolites or whether they are secondary to other changes which these very reactive molecules can induce in vivo. Evidence over the last few years now indicates that the cytoskeletal proteins may constitute direct molecular targets for the oxons. Indeed, CPO has been shown to be able to bind to and covalently modify (organophosphorylate) some important cytoskeletal proteins including the core proteins of the MT and MF network, tubulin and actin. The extent of potential CPO reactivity is demonstrated by its capacity to bind covalently not only to serine but also to tyrosine and, even, to lysine residues of these proteins. Thus, in vitro, CPO can covalently link to five lysine residues of purified bovine actin [66]. In the case of tubulin, which has been studied in more detail, CPO can bind to no less than 17 tyrosine [67] and five lysine [65] residues under in vitro conditions. However, these experiments have adopted the use of some very high (up to 0.5 mM) concentrations of the oxon, and when CPO has been employed at concentrations that are biologically relevant, that is, in the low micromolar range, only one tyrosine residue of purified tubulin (Tyr 83 of a-tubulin) becomes organophosphorylated [67]. More importantly, in vivo treatment of mice with six sublethal doses of CPO over a

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period of 50.15 hr leads to the organophosphorylation of brain tubulin on residue Ser 338 of b-tubulin [48]. This residue is not found labelled after in vitro incubation of purified (MAP-free) bovine tubulin with CPO [67]. Finally, treatment of mice with a non-AChE-inhibiting dose of CP for 14 days results in the binding of CPO to residue Tyr 281 of b-tubulin, the tyrosine residue previously found to be the most reactive also amongst the eight tyrosine residues of b-tubulin in vitro [48]. Summary, Additional Remarks and Conclusion Amongst the various classes of OP insecticides, the phosphorothionates rightly are the focus of toxicological interest, as they encompass a large number of compounds several of which are heavily involved in poisonings around the globe. Central to the metabolism of phosphorothionates in vivo is their conversion to their oxon analogues. The toxicological implications of this biotransformation are becoming increasingly clear, as the oxon metabolites formed seem to be guilty of inducing a number of distinct neurotoxicities. Evidence over the last 15 years indicates that the oxons may be capable of inducing developmental neurotoxicity, as a result of their ability to target separately several stages of the neurodevelopmental process. Due to their high reactivity, the oxons have been found to target, apart from AChE, several compounds, but the relevance of many of these targets to neurodevelopment is obscure. The possibility of the neuronal cytoskeleton being a target for the oxons (and, possibly, other developmental neurotoxicants) makes theoretical sense because of the known critical importance of the cytoskeletal proteins in the various stages of normal neurodevelopment. The neuronal cytoskeleton is a good target for the oxons also because several neurodevelopmentally important cytoskeletal proteins contain in their molecule an unusually high number of serine residues and, thus, sites suitable for attack and phosphorylation by the oxons. Actual experimental data obtained over the last 5 years and reviewed here do indeed indicate that the oxon metabolites of phosphorothionates, and particularly CPO and DZO, at concentrations that can be found in real life, exert potent and wide-ranging effects on the neuronal cytoskeleton. After our very recent studies on the effect of DZO on the actin-binding protein cofilin, it appears that the oxons may disrupt all three components comprising the neuronal cytoskeleton. This disrupting action is expressed at various levels, affecting cytoskeletal protein levels, intracellular distribution, post-translational modification, dynamics and function. The use of differentiating neuronotypic and gliotypic cell lines in several of these studies has been important in demonstrating that the oxons’ disrupting effects may involve the cytoskeleton of both neuronal and glial cells and that the effects on these two types of cells are not identical. These effects are not secondary to other changes, but they represent primary, direct effects of the oxons on core proteins of the cytoskeleton, with CPO covalently binding and organophosphorylating multiple sites on both tubulin and actin. Although the use of realistic oxon concentrations markedly reduces the number of amino acid residues of tubulin that become organophosphorylated

and the identity of these residues seems to vary according to the dosage regimen, the obtained data do indicate that welltolerated doses of oxons can target and organophosphorylate both serine and tyrosine residues of tubulin. On the other hand, endogenous phosphorylation at these sites has not been reported, and thus, the functional implications of their organophosphorylation by the oxons are not clear. Conduction in the future of analogous experiments with cytoskeletal proteins containing a much higher than tubulin number of serine residues, for example NFM and NFH, would be important for demonstrating the phosphorylating capacity of the oxons and could reveal organophosphorylated sites whose endogenous reversible phosphorylation is relevant to neurodevelopmental function. Although altered phosphorylation of cytoskeletal proteins can be the result of their direct binding by the oxons, it may also arise as a result of the oxons targeting the various cell signalling pathways known to regulate the phosphorylation of the cytoskeletal proteins. The ability of CPO to directly bind adenyl cyclase and diacylglycerol lipase implies that this oxon may target important signalling pathways. Interestingly in this respect, recent, unpublished data from our group show that MAP kinase-ERK 1/2, which phosphorylates a number of cytoskeletal proteins and is a major convergence point for other developmentally important signalling pathways [68], is severely affected in differentiating N2a cells by DZO. In conclusion, the developmental neurotoxicity induced by the phosphorothionate insecticides may, amongst others, be due to the ability of the oxon metabolites to disrupt the neuronal cytoskeleton, partly because these substances have the capacity to multiply bind and covalently modify by phosphorylation a number of cytoskeletal proteins that are essential for normal neurodevelopment. The data show emphatically the extensive range of neurotoxicities and neurotoxic targets associated with the oxon metabolites. In this context, the data enhance the importance of the cytoskeleton as a target in the toxicological action of chemical compounds. In addition, they clearly indicate that the oxons are not merely AChE inhibitors. Rather, these compounds have the capacity to attack a number of neurotoxic targets and induce a range of neurotoxic effects, some of which may cause harm very early in life. Acknowledgements The work of the author and associated research collaborators on diazoxon and diazinon in N2a cells was supported by grants from Pfizer Ltd. and the Department for Environment, Food and Rural Affairs (defra), UK, respectively. References 1 Kobayashi H, Suzuki T, Akahori F, Satoh T. Acetylcholinesterase and acetylcholine receptors: brain regional heterogeneity. In: Satoh T, Gupta RC (eds). Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Wiley, Hoboken, NJ, 2010;3–18. 2 Abou-Donia MB. The cytoskeleton as a target for organophosphorus ester-induced delayed neurotoxicity (OPIDN). Chem Biol Interact 1993;87:383–93. 3 Quistad GB, Barlow C, Winrow CJ, Sparks SE, Casida JE. Evidence that mouse brain neuropathy target esterase is a lysophospholipase. Proc Natl Acad Sci USA 2003;100:7983–7.

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