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High Dose

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Scientific papers - High dose - page 6

Phosmet metabolism

Phosmet generally metabolizes 'in vivo', via a series of toxic intermediates, into methylsulfonyls, methylthio-phosphoric acid, phthalimide, oximethylphthalimide, chloromethylphthalimide/00-trimethylphosphorodithioate (as impurities (42)) and formaldehyde (34).

As the phthalimido moiety is unique to the phosmet type of OP compound, and the high-dose usage of systemic phosmet is unique to the UK, it would seem that the phthalimido moiety is the most likely hypothetical candidate for the responsibility for initiation of PrPbse. But, in order to find, by elimination which phosmet moiety or moieties are singly or jointly I active' in transforming PrP into the abnormal form in these treated cells, it would be necessary to conduct several trials where PrP cells are exposed to pure phthalimides or oxones as well as to other types of OP which lack the phthalimido, sulphur moieties, etc.

Considering the diverse range of phosphorylating, acylating and alkylating covalent effects that various types of OP and their respective toxic metabolites are recognized to exert directly upon various membrane proteins and indirectly upon various cytoskeletal proteins, a multitude of feasible, 'exotic' mechanisms can be speculated when it comes to gauging the pathways of interaction between phosmet and PrP, and its hypothetical initiation of PrPbse.

Given that phosmet and other 0Ps have been shown to integrally interact with so many target sites and metabolic pathways (such as nerve growth factor receptors (59-61), T-cell activation (24), calcium channels (62,63), GABA-mediated channels (62,64), phospholipase C (65,66), membrane phospholipids (5,34,66)) with which prion protein is also metabolically involved (7,23,28,67,68), it is not surprising that the results of these trials at the Institute of Psychiatry have demonstrated an interaction between PrP and phosmet.

The symptoms of chronic OP poisoning in cattle duplicate those observed in BSE (2,69), indicating, at the very least, that chronic OP intoxication targets and disturbs the same CNS tracts as are lesioned in BSE. Rare cases of OP poisoning have produced spongiform encephalopathy in certain contexts (70), but as there is a total absence of any transmission studies in the literature where healthy animals have been inoculated with brain homogenate taken from these OP fatalities, one can only assume that no attempts have been made to transmit these OP induced SEs.

Mechanism 1: Phosmet interaction with the phosphatidylinositol glycolipid anchor that is conjugated onto PrP, and its subsequent disruption of PrP tertiary folding and the phosphoinositide second messenger signal transduction cycle (see Fig. 2)

It is proposed that phosmet induces a two-stage interaction with the P1 glycolipid anchor either prior to or during its conjugation to PrP at the ER (71), or whilst anchored to the external surface of the neuronal membrane.

Phosmet and other 0Ps have been shown to bind and concentrate in the phospholipid phase of membranes (5,34,66), interacting with phospholipids such as PIS causing an alteration to the structure, function, and activity of the membranes and their membrane associated enzymes/proteins - which would include PrP.

Thus, the P1 membrane lipids of the OP poisoned individual would act much as a 'toxic bank' of OP-contaminated lipids that are capable of 'infecting' various membrane proteins (such as PrP) that are programmed to conjugate with PIS to form anchors onto the external surface of nerve membranes.

The OP-modified PI anchor 'infects' the protein after conjugation with the anchor at the ER. This would affect the allosteric behaviour and final folding of the main protein body. One study suggested that such a 'knock-on' effect occurs with acetylcholinesterase (one such membrane protein that conjugates onto PIS (71)) following OP modification of its P1 anchor (72).

Research has also shown that OP intoxication causes a redistribution of these phospholipids from the inner to the outer monolayer of the plasma membrane (73)

It is proposed that phosmet modifies an endogenous phosphorylation site (as proposed in my previous paper (2) as serine 231) found on the PI anchor of bovine PrP. For PI is well recognized to accommodate such an endogenous phosphorylation site for protein kinase C (74). This site fulfils a critical link in the phosphoinositide cycle where PI is phosphorylated into phosphatidylinositol-4 5-bisphosphate. OPs have also been demonstrated to block phosphorylation of this site in the context of PI found on sperm membranes (75), thus providing strong grounds for suggesting that OPs must exert the same impact on the PI anchor conjugated onto PrP.

Serine 231 is markedly bipolar (76); one side of the disk is negative and the other positive, which enables the prion protein to anchor itself electrostatically to a positively charged phospholipid head on the membrane, e.g. PrP's PI anchor - which then orientates the negative face of the prion protein outwards.

Such a phosmet modification at serine 231 would leave an extra negative charge that disrupts the electrostatic equilibrium of this bipolar site, causing, amongst other disturbances upon conformational development and allosteric properties, a deflection of protein kinase C away from its preferred phosphorylation site so that it phosphorylates the anchor (or perhaps a site on the main body of PrP itself) at an alternative site, invoking other normal PrP isoforms to abnormally phosphorylate likewise (77). The net effect would produce a cascade of abnormally phosphorylated PrP which initiates a long-term escalation of pathogenic consequences in a multitude of metabolic directions.

Low doses of OPs such as parathion (12,78) have been shown to activate turnover of the entire phosphoinositide second messenger cycle, via an OP-induced increased turn over of acetylcholine at the muscarinic cholinergic receptors. This activates a G protein which, in turn, stimulates hydrolysis of the phosphatidylinositols by phospholipase C, causing a build-up of inositol phosphates, which dramatically modulates signal transduction in the cell. For inositol phosphates are hydrolysed into two intracellular signals - inositol trisphosphate, which triggers the release of calcium, and diacylglycerol, which stimulates the activities of the phosphorylating protein kinase C.

Changes in protein kinase C have been repeatedly noted in many studies where neuropathic OPs (79), like phosmet, have been administered into animals. The net effect of their elevated level is to modify various cytoskeletal proteins at high-affinity binding sites which can invoke neurodegenerative sequelae (21), as well as exerting feedback effects on several aspects of the phosphoinositide cycle (12). It is proposed that the OP-induced increased turnover of the protein kinase C invokes a hyperphosphorylation at a displaced abnormal site on PrP's PI glycolipid anchor or on the main body of PrP itself.

This OP-induced 'vicious circle' of overdrive of the phosphoinositide signal transduction cycle can have other major repercussive effects upon neurotransmission, including blocking the inhibitory action of several transmitters such as serotonin (74), a recognized phenomenon of prion disease (80).

One of the most severe pathogenic repercussions of overdriving the phosphoinositide cycle - coupled to the OP-induced upregulation of the WDA glutamate receptors (15) - must lie with the increased intraneuronal influx of calcium, which, in turn, causes the excessive release of nitric oxide free radicals into the neurone (16). As OP intoxication has demonstrated effects of peroxidation of various phospholipids (17) by upregulation of superoxide dismulase, catalase, etc., it could be postulated that this OPinduced cascade of free radicals in neurones could 'infect' and interact with essential electron acceptor sites on the polar heads of the PI anchor membrane lipids or perhaps with the copper or tyrosine free radicals (81) on the main body of PrP itself. Initiation of such a transformation of copper transition domain on PrP into the highly reactive copper free radical could account for the transformation of PrPc into its misfolded, 'infectious' isoform. Thus once PrP is 'infected' with such reactive radicals, the chain reaction of the prion disease process can ensue.

Interestingly, abnormal PrP function in prion disorders has been linked to a breakdown in calcium homeostatis resulting in apoptosis and disruption of cell signalling via calcium-mediated neurotransmitter release (28,29), which lends weight to the suggestion that an underlying disturbance in the phosphoinositide cycle is centrally involved in the pathogenesis of BSE and other TSEs. In order to test this possibility, BSE-suffering cattle should be treated with the pharmaceutical 'neomycin', which is known to inhibit activity of various subtypes of phospholipase C, thus blocking the entire phosphoinositide cycle. Response of the BSE cow to neomycin therapy might well provide a good indication of the status of the phosphoinositide cycle during the various stages of BSE pathogenesis.

It is the action of phospholipase C on the PIs in the membrane and glycolipid anchors that causes the release of DAG, which transduces signals across the plasma membrane (74). Interestingly, one of the abnormal characteristics of the TSE prion in relation to the normal PrP isoform is that it fails to detach from its anchor within the normal time span (68), suggesting that some aspect of PI specific phospholipase C cleavage has been impaired. In BSE, it is proposed that phosmet has covalently and/or structurally modified the anchor so that phospholipase C can no longer access its catalytic site properly, although the other subtypes of phospholipase C may well continue to hydrolyse their phospholipid substrates effectively, thus enabling the continued increased turn over of G protein signalling.

To confuse an already multi-complex issue, high doses of some specific types of serine esterase inhibitors such as 0Ps have been demonstrated to block one of the four subtypes of phospholipase C which cleaves the PI glycolipid anchors, as well as block the resulting mobilization of arachidonic acid (65). Other research demonstrates that 0Ps inhibit phosphatidylinositol phosphodiesterases (66), which is perhaps achieved either via a direct OP covalent phosphorylation of its serine esterase site, or by the alkylating activities of some OPs, like phosmet (6), modifying the activity of guanine nucleotide binding proteins which regulate turnover of these phosphodiesterases.

The overall long-term disruptive impact of 0Ps on the homeostasis of the phosphoinositide system can only lead to a multicomplex, multisite disturbance of the second messenger signalling system with widespread repercussions via disturbance of voltage sensitive calcium channels and membrane permeability, lipid peroxidation due to the generation of nitric oxide free radicals (16,17), etc. where important calcium and protein kinase C-mediated feedback is corrupted into creating a cascade of abnormal hyperphosphorylation of cytoskeletal and membrane proteins such as PrP, tau and neurofilament proteins, etc.

Thus, if a PrP PI anchor's phosphorylation site has become covalently modified by OP in the first instance, it is feasible to envisage a two-stage toxicologlcal process operating in tandem; where a simultaneous OP activation of the phosphoinositide cycle would lead to increased feedback of protein kinase C on some alternative site on PrP or its PI anchor. where the kinase C has been deflected from its normal phosphorylation site due to a prior OP modification. This results in the phosphorylation of the protein at an alternative site, which initiates a chain reaction, whereby the abnormally phosphorylated PrP induces other normal PrP isoforms (undergoing final constitution at the ER) to abnormally phosphorylate likewise (77); creating an everincreasing contagious cascade of abnormal protein transformation.

The end result leaves an abnormal distribution and intensity of negative electro charge on the surface of PrP. This charge upsets electrostatic equilibrium and/or the Van der Waals forces within the folding protein disrupting the final stages of tertiary folding of PrP, perhaps blocking the cleavage[bonding sites of isomerases/foldases. The resulting misfolded PrP isoform invokes chaperone stress proteins to conjugate onto it (26), thus blocking some of the proteolytic cleavage sites rendering misfolded PrP partially resistant to protease degradation.

Auto-antibodies (24,25) could also be raised against the resulting phosmet-PrP-chaperone complex producing an unconventional 'non-inflammatory' pathology characteristic of TSEs due the abnormal prion's inability to perform its normal PrPc role (23) of activating lymphocytes and the resulting inflammatory response.

It is possible that the aetiology of several neurodegenerative diseases such as motor neurone disease and Alzheirner's disease, which involve the deformation and mutation of various membrane/cytoskeletal proteins in the early stages of their pathogenesis, could also partially hinge upon the modification of phospholipid membrane anchors by a whole range of exogenous metals and organo-pollutants present in the early life environment of the victim.

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Last updated 09-Feb-2007