Back to Purdey Home Page   

Medical Hypotheses (1998) 50, 91-111 c.Harcourt Brace & Co. Ltd 1998

High-dose exposure to systemic phosmet insecticide modifies the phosphatidylinositol anchor on the prion protein: the origins of new variant transmissible spongiform encephalopathies?

M.PURDEY

High Barn Farm, Elworthy, Taunton, Somerset, TA43PX, UK

Abstract - Compulsory exposure of the UK bovine to exclusively high biannual doses of a, 'systemic' pour-on formulation of an organo-phthalimido-phosphorus warblecide, phosmet, during the 1980s (combined with exposure to the lipid-bound residues of 'bioconcentrated' phosmet recycled back via the intensive feeding of meat and bone meal), initiated the,'new strain' modification of the CNS prion protein (PrP) causing the UK's bovine spongiform encephalopathy (BSE) epidemic. A lipophilic solution of phosmet was poured along the bovine's spinal column, whence it penetrated and concentrated in phospholipids of the CNS membranes, covalently modifying endogenous phosphorylation sites on phosphatidylinositols (PIs) etc., forming a 'toxic membrane bank' of abnormally modified lipids that 'infect' any membrane proteins (such as PrP) that are programmed to conjugate onto them for anchorage to the membrane. Thus, phosmet invokes a primary covalent modification on PrP's PI anchor which, in turn, invokes an overall diverse disturbance upon CNS phosphoinositide second messenger feed back cycle, calcium homeostasis and essential free radicals; thus initiating a self-perpetuating cascade of abnormally phosphorylated PI-PrP that invokes a secondary electrostatic and allosteric disturbance on the main body of PrP impairing tertiary folding. Chaperone stress proteins conjugate onto misfolded PrP blocking its sites of proteolytic cleavage. Fresh epidemiological evidence is presented and experimental evidence referenced that adds support to a multifactorial hypothesis which proposes that BSE is a hitherto unrecognized and previously unmanifested class of subtle chronic phosmet-induced delayed neuro-excitotoxicity in the susceptible bovine.

Received 13 May 1997 Accepted 12 June 1997

Introduction

The aetiology of the familial and sporadic types of prion disease, such as sporadic Creutzfeldt-Jakob(CJD) and scrapie, is considered to hinge, in part, upon a diverse range of abnormal isoforms of a host encoded glycoprotein called prion protein (PrP) (1). On the other hand, BSE and 'new variant' CJD (nvCJD) are characterized by one uniform isoform of abnormally modified PrP (1), which suggests the presence of some hitherto unidentified common environmental trigger factor that was initially introduced into the bovine environment at some point during the late 1970s/early 1980s.

Further research continues to support the hypothesis (2) that. the UK's exclusive mode of compulsory high-dose usage of a systemic formulation of phosmet (N-mercaptomethyl-phthalimide, S-00-dimethyl phosphorodithioate) insecticide directly upon cattle for warble fly eradication during the late 1970s/1980s, coupled to the recycling of bioconcentrated phosmet in the tallow fraction of cattlefeed, was the primary trigger that initiated the abnormally modified prion protein, causing the onset and spread of the UK's 160,000 -case BSE epidemic.

CNS penetration by systemic organophosphorus compounds

Organophosphorus (OP) warble fly treatments (such as phosmet) are manufactured in a lipophilic, 'systemic' formulation that enables the active ingredients to penetrate through the hide and internal body membranes, permeating the whole internal environment of the bovine so as to exterminate any warble parasite present. The OP was applied in a 'pour-on' solvent-based oil solution, applied along the back of the head and the entire length of the spinal column.

Cattle very occasionally developed a paraplegic syndrome as an acute abnormal reaction to OP warblecide treatment (3,4). This was usually caused by an anaphylactic-type reaction to the dead warble larvae that had been intoxicated by OP whilst congregating in the epidural fat of the spinal cord during the later stages of their annual life cycle. Such complications following use of OP warblecides were largely avoided because treatment during the winter months was prohibited - the stage of the life cycle when warbles could sometimes be found inside the spinal canal.

The fatal intoxication of warbles in the spinal canal following treatment with recommended doses of systemic OP warblecides demonstrates that 0Ps are penetrating the phospholipid membranes in the spinal canal at a dose which is lethal to the warble.

'Toxic phospholipid membrane bank' - the infectious vehicle in transmissible spongiform encephalopathies.

Phosmet and various other 0Ps are well recognized to bind and concentrate in the phospholipid phase/ liposomes of neuronal membranes in the CNS (5,6) causing an alteration in the charge of the polar heads of the affected phospholipids. This disrupts the overall structure, function and activity of these membranes with 'knock-on' effects on the allosteric behaviour/folding of their membrane-associated proteins; such as PrP. Interestingly, PrP is found anchored/conjugated to the same types of phospholipid (7), phosphatidylinositol (P1), in the nerve membranes which are targeted by phosmet, and PrP is recognized to form phospholipid-protein complexes in liposornes (7) found in the membranes of select tracts in the CNS.

Thus, intoxication of the organism with a toxic membrane bank of phosmet-modified phospholipids is subsequently capable of 'infecting' any membrane associated proteins - such as PrP - that conjugate onto them.

If phosmet does indeed initiate the 'infectious' new variant PrP, then the 'systemic' route of chemical entry after its delivery along the spinal column/ headline perhaps explains why brain titrations/tissue assays in clinical cases of BSE have detected infectivity exclusively in brain and cervical spinal cord (8 [p. 64]). By contrast, in scrapie and chronic wasting disease (CWD) (the prion disease found in cervidae), infectivity originates in the tissues of the gut and lymphoreticular system and then travels to the CNS over a 2 year period - suggesting the causal agent enters via the oral route in scrapie/CWD.

It could also be suggested that phosmet will only covalently modify and unleash pathogenic consequences of multireplication in a specific subtype of PrP which is expressed exclusively within the CNS and implicated in the aetiology of nvCJD and BSE (1); this explains why the residues of phosmet, which are complexed with the lipid/fat fractions of meat and bone meal (MBM) and are recycled back into the cow via the oral route, still fail to modify the cellular prion protein (PrPc) subtypes that are expressed in spleen and the lymphatic system.

Furthermore, the specific CNS P1s to which phosmet initially complexes following exposure to systemic pour-on treatment, may well demonstrate specificity towards conjugation with PrPc subtypes that are exclusively expressed in CNS cell lines; this explains why any phosmet-phospholipid complexes that are recycled back into the bovine as residual contaminants in MBM feed are only able to covalently conjugate with the PrP subtypes implicated in BSE/nvCJD and exclusively confined to the CNS.

Phosmet-induced covalent modification of prion protein

It is hypothesized that the high dose OP warblecide phosmet induces a hitherto unrecognized abnormal irreversible post-translational modification of CNS PrP, most probably on its PI glycolipid anchor. This impairs tertiary folding of PrP, resulting in the formation of a conformationally deranged isoform of PrP. Thus, the biochemical genesis of prion formation involves a two-stage process, implicating a primary covalent modification of PrP which causes a secondary conformational modification of PrP during the tertiary folding process.

Phosmet is classed as a 'neuropathic' type of OP (9). Like other types of OP in this class, high doses of phosmet have been shown to modify certain membrane proteins such as neurotoxic esterase by inducing a covalent modification which produces an irreversible conformational change (10) via both direct phosphorylation/aging and alkylation of various active sites (6,11). It is therefore feasible to propose that phosmet could also directly modify the conformation of prion protein and/or its glycolipid anchor in some way, or perhaps indirectly, by influencing other proteins, etc. which are integral to the activities of PrP in cells. For instance, subacute low doses of 0Ps such as phosmet can disrupt turnover of the phosphoinositide second messenger cycle (12) which is interlinked to the activities of PrP and its PI anchor, or phosmet can inhibit glycosidases (13) which could impair/block deglycosylation at linked glycosylation sites on PrP (14). 0Ps such as phosmet can overexcite NMDA receptors (15) which would generate free radicals (16) that peroxidize membrane lipids such as PrP's PI anchor or disrupt essential electron interactions with tyrosine radicals in the main body of PrP. The further effects of OP increased turnover of glutathione peroxidase, superoxide dismutase (17) or cytochrome P450 activity would further aid the proliferation of neurodegeneration created by the OP-induced generation of free radical chain reactions.

Hypothetically, once PrP is abnormally modified and 'aged', the extra negative charge corrupting PrP's molecular surface subsequently disrupts interaction with foldases/isomerases or Van der Waal's forces during tertiary folding, which could lead to an influx of chaperones that bond onto misfolded PrP, blocking proteolytic/phospholipase C cleavage sites, causing the development of the new variant misfolded PrP isoform.

Whilst potential sites for phosphorylation on PrP have been researched by Stanley Prusiner's team (18), the characteristic acid-labile nature of these sites made such an investigation impossible to conclude due to the acidic conditions employed in the highperformance liquid chromatography purification procedure. This work did not recognize and take into account the well-recognized protein kinase C endogenous phosphorylation sites on the phosphatidylinositol glycolipid anchor that is conjugated onto the C terminus of PrP. However, studies investigating the activities of phosphorylating and dephosphorylating enzymes in the TSE diseased brain have demonstrated abnormally elevated levels (2), suggesting that some aspect of phosphorylation may play a role in the pathogenesis of the disease.

Like other neuropathic 0Ps, phosmet can covalently modify and cause conformational changes upon a diverse array of membrane and cytoskeletal proteins such as neurofilament protein, tubulins and microtubule-associated proteases (10,19-22). Some of these proteins are modified indirectly, by the secondary messenger 'feedback' mediator protein kinase C as a result of the OP's disturbing turnover of the phosphoinositide cycle (see biochemical section) or by the OP and/or calmodulin kinase 2's phosphorylating high-affinity neurotoxic binding sites (21).

An unconventional autoantibody binding onto the phosmet modified prion protein-chaperone complex (or onto other types of modified membrane proteins) could also set up the final stages of pathogenic complications creating the so-called 'infectious' protein end product of the prion disease process. The pathology of conventional autoinimune diseases is characterized by inflammation and mononuclear infiltrations, but the lack of the inflammatory response in TSE pathology could be attributed to the failure of the abnormal prion protein to perform its normal function in the pathways of lymphoid activation (23). Thus, phosmet-modified brain protein is then artificially inoculated into a healthy, uncontaminated secondary host, then that organism will mount its own autoantibody assault against the exogenous challenge, which, in turn, creates spongiform degeneration.

Several studies have demonstrated how autoantibodies are raised against OP-protein modified complexes in humans who have been occupationally exposed to low doses of these chemicals (24,25).

Interestingly, analysis of the prion rods that hallmark the CNS pathology of BSE brain reveals that prion protein is not the sole component; for the microtubule-associated protease, 'tau', and 'neurofilament protein' (8), as well as other unidentified tightly bound proteins (26) (possibly chaperones) are also found in association with PrPbse in these rod structures. The presence of these other proteins besides PrP does not necessarily imply that they represent mere incidental artefacts of the prion disease process, as some researchers suggest. They may also be fulfilling some hitherto unrecognized primary or secondary aetiological input into the pathogenesis of prion disease, albeit without unleashing the same devastating degree of 'infectivity' and pathogenic propensity to initiate long-term delayed neurotoxicity as has been attributed to the abnormal PrP isoform.

However, recent French research (27) has demonstrated that TSEs can be artificially transmitted into the recipient animals whose resulting CNS pathology fails to demonstrate the presence of prions at post mortem. This could suggest that some particle or site on other membrane/cytoskeletal proteins implicated in TSEs could also carry an 'infectious' pathogenic property (e.g. a phosmet-induced abnormal charge or free radical) in common with the 'infected' prion, such as the OP-modified PI anchor complex. Once injected into the healthy organism, that OP-modified PI is then capable of conjugating onto other types of membrane protein besides prion protein, thereby invoking a similar mode of steric hindrance/ misfolding in the main body of those proteins which manifests similar aspects of pathology and symptomology that are expressed in true prion disease.

'BSE positive', whose CNS pathology is characterized by prion rods, amyloid plagues and spongiform degeneration, could implicate an aetiology of 'in utero' phosmet intoxication where abnormal isoforms of both PrP and various other hitherto unrecognized delayed neurotarget proteins are implicated in the pathogenesis. On the other hand, 'BSE negative', whose pathology is neither characterized by prion rods nor spongifonn degeneration, involves an aetiology of 'postnatal' phosmet intoxication where various types of neurotarget protein are abnormally modified - and conceivably rendered 'infectious' - yet PrP is spared. One could also argue that 'BSE negative' represents the phosmet or other OP-poisoned individual who carries a PrP genotype which does not express susceptibility to prion disease.

Extremely high doses of these neuropathic OPs, as ingested during a suicide attempt or accidental poisoning, have produced a characteristic irreversible neuropathy in such misfortunate victims. It is proposed that when lower, subacute doses of the unique phthalimido-formulated neuropathic OP, phosmet, is absorbed by the CNS (perhaps during embryogenesis), then a hitherto unrecognized subtle modification to the prion and other membrane/cytoskeletal proteins is initiated. Once other TSE promoting secondary cofactors come into the play, a cascade of infectious prions is created. These partially proteaseresistant prions and associated proteins start accumulating over the 'so-called' incubation period until they reach critical pathogenic levels; whence outward symptoms of disease start to manifest years after the initial trigger event.

The characteristic spongiform pathology of BSE is caused, in part, by the failure of the misfolded. prion to perform its normal regulatory role at specific voltage-sensitive calcium channels, which leads to a rise in intracellular free calcium, resulting in the generation of nitric oxide free radicals (16), apoptosis and cell death (28,29). An unconventional autoantibody assault on the phosmet modified PrP-chaperone complex may ensue, whereby a tightly bound antibody attaches to modified PrP, impairing tertiary folding. Pathology lacks the usual inflammation characteristic of autoimmune disease because the abnormal prion is no longer capable of performing its normal role of activating lymphocytes (23).

The specific CNS distribution of the spongiform pathology may also be partly reflected by the failure of the misfolded. prion to perform its assumed role in transportation of copper ions to the copper-dependent neuronal systems, such as the dopaminergic tracts. Copper deficiency in these regions would further inactivate copper-dependent superoxide dismutase leading to the further accumulation of neurotoxic free radicals. Interestingly, both of the copper chelating chemicals, cuprizone (30) and disulfiram (31) produce a 'short circuited' type of 'pseudo' SE without having to exert a direct modification upon PrP's copper carriage sites. Interestingly, a high prevalence of SE in humans occupationally involved with disulfiram in the vulcanizing process of auto tyre manufacture has been reported (31).

Spatiotemporal epidemiological correlations between phosmet use and BSE incidence

The UK was unique in its use of systemic phosmet in that it compelled farmers to apply the chemical biannually at a 20 mg/kg dose rate recommending an optional 10 mg/kg follow-up dose 14 days later. By contrast, the few other countries that have licensed phosmet in its systemic pour-on formulation have only licensed it for voluntary treatment of lice in cattle and/or pigs as a 'one off' dose of 10 mg/kg or 2 mg/kg respectively.

Treatment of the UK cattle herd with systemic phosmet became particularly intensive during the early 1980s, when compulsory biannual treatment was instigated in 1982 (2). And then in 1985, dairy farmers had no choice but to apply the phosmet type of warblecide because the use of all the other competing types of OP warblecide became restricted following alterations in licensing criteria.

Whilst the warble fly was largely eradicated from the UK by the end of the 1980s, very small pockets of infestation still occasionally emerge. Cattle residing in these warble-infested zones, as well as all cattle that are importect into tne ut,.-, are stin supjectecl to compulsory treatment with systemic phosmet at the 20 mg/kg warble dose rate.

There is also some usage of systemic phosmet for the voluntary control of lice and mange, etc. in cattle and pigs. Cattle are treated with a 10 mg/kg dose which is followed up by a further 10 mg/kg dose 14 days later, e.g. still 20 mg/kg in total.

Such a substantial drop in phosmet usage since the peak of the warble eradication era in the mid 1980s, coupled to the complete removal of tallow/animal fat from the bovine food chain in 1995, could well account for the substantial down turn in BSE incidence in the UK since 1994.

This theory postulates that various 'optimum' prerequisites surrounding phosmet usage must be fulfilled before BSE can emerge at a significant incidence rate. They are as follows :

1 . Phosmet must be applied onto livestock in a ,systemic' lipophilic pour-on formulation, e.g. enabling the active ingredients to penetrate through all membrane barriers and bind into adipose and phospholipids (5,6) - such as the PI fractions of the CNS nerve membranes to which the prion protein is anchored.

2. Phosmet must be applied twice annually at its high dose rate of 20 mg/kg (with 10 mg/kg 14 day repeat) as was compulsory during the UK's warble eradication programme.

3. Phosmet exposure must take place during early life/in utero stages of the animal - NB, either via direct systemic phosmet treatment and/or indirectly via ingestion of concentrated feeds containing bioconcentrated phosmet in fat derived from rendered-down pigs or cattle.

4. BSE will more likely affect an animal that is genetically or environmentally predisposed to less efficient detoxification of phosmet and to susceptibility to prion disease.

The European/global perspective on phosmet-induced BSE - a comparative study of phosmet used and BSE incidences in various different countries

Apart from the UK, no other country licensed systemic phosmet for veterinary use whilst adhering to the aforementioned 'optimum prerequisites' of intensive exposure. (This information was obtained through communications with the various veterinary pesticide licensing bodies of each country.) However, Eire, the Channel Islands, France and Switzerland appear to be the only other countries that have partially satisfied these prerequisites for BSE onset by exposing their livestock (cattle and/or pigs) to direct applications of systemic phosmet - albeit voluntarily and at relatively lower doses - as well as to feedingstuffs containing the recycled lipids of livestock treated with phosmet.

Apart from Portugal which only exposed its cattle to an in-feed source of bioconcentrated phosmet/ prions in the fat/tallow fraction of MBM imported from the UK, the only other countries affected with endemic BSE outside of the UK are Eire, the Channel Islands, France and Switzerland which are the other countries besides the UK that have exposed their livestock both directly and indirectly to potentially significant doses of phosmet.

Eire first took up the use of systemic phosmet for controlling warble at the end of their warble-fly eradication programme in 1978 (32). But phosmet use was confined to one district of Eire only, where it was applied once annually as an autumn dressing and used at a low dose rate of 6 mg/kg bodyweight (32) - a fivefold lower dose than the 20 mg/kg (plus 10 mg/kg 'follow up') biannual dose employed in the UK.

After warbles were successfully eradicated in Eire, systemic phosmet was still used voluntarily for controlling lice and mange. The dose rate for lice was increased around 1992 in Eire to the same dose rate as applied in the UK, 10 mg/kg, with a repeat treatment recommended for 14 days later. Allowing for the 4-year average incubation period of BSE in cattle, the recent five-fold increase in the annual BSE incidence rate in Eire in 1996 over previous years (Eire Dept of Agriculture) could perhaps be explained by the increase in Eire's phosmet dose in 1992.

Australia and New Zealand are the only countries that have licensed systemic phosmet for veterinary use and have not reported BSE. But their usage on cattle was voluntary and confined to the control of lice at a 10 mg/kg low dose without the 14 day repeat treatment, which constitutes a considerably less intensive application than applied in the UK. There is also little possibility of a source of in-feed phosmet (within MBM) bioconcentrating into calves/cattle in the extensive grass-fed livestock systems practised in these countries. For an almost negligible input of concentrate feeds containing MBM are recycled back into cattle in these countries compared with the intensive reliance upon the recycling of MBM on UK farms prior to 1988 The New Zealand Meat Industry Association reports that 60% of its MBM is exported to Asia, with the remainder being fed to poultry and pigs. The same pattern of MBM distribution is applied in Australia.

Phosmet has also been employed for use on cattle in the USA at a much reduced dose of 2 mg/kg as a non-systemic, back rubber/powder/spray-on 'contact' type of formulation where the chemical is designed to control ectoparasites, and fails to penetrate through the hide of the cow and gain access to the CNS.

US pesticide licensing authorities deem that milk taken from cows that have been treated with this relatively low-dose formulation of phosmet has to be withdrawn from sale for human consumption for a period of 28 days following treatment (in contrast to 6 h in the UK). This measure served to debar the use of phosmet upon milking cows in the USA for economic reasons. It also had the effect of excluding cattle in their first 6 months of pregnancy when they are in milk from exposure to phosmet, thus avoiding any possibility of vulnerable 'in-utero' exposure to this chemical - a developmental stage when PrP could be more susceptible to phosmet modification and the fetus being a target of phosmet concentration.

An interesting comparative study into the different modes of controlling warble fly in various different European countries, by A. Liebisch (33), demonstrates how the UK adopted a far greater degree of 'chernical intensity' in relation to other countries by employing compulsory measures to eradicate warble flies. Such measures enforced biannual treatments of high-dose systemic 'pour-on' 0Ps as opposed to the lower dose or 'spot-on' types of 0Ps (which used considerably less active ingredient per treatment) used by some other countries as a single annual application only.

Apart from the UK, Eire, France and Switzerland, which have adopted compulsory warble control measures, other European countries have adopted voluntary or non-existent measures, which perhaps explains why no other country apart from Eire and the UK has so far succeeded in eradicating the fly.

Liebich's study also demonstrates that phosmet was not recommended for use upon cattle in any other country's compulsory warble control programme operating outside of the UK (apart from some very limited use in Eire and France during the late 1970s). This explains why no correlation exists in the greater majority of BSE endemic countries outside of the UK linking compulsory warble treatment zones with the spatiotemporal distribution of BSE incidence. By contrast, in the UK, where phosinet was employed at high systemic doses, a general correlation does exist (2).

Various countries outside the UK which have attempted to control the warble fly, such as Switzerland, Denmark, France (33), USA, Canada, have all employed non-phosmet types of systemic 0Ps such as trichlorphon, famphur, cournaphos - or the non-OP ivermecti.n. None of these compounds contains the phthalimido moiety that is found exclusively within the phosmet compound. In fact,

Phosmet is the only OP compound and systemically formulated pesticide to contain a phthalimido moiety.

Some of the toxic metabolites that derive from phosmet parent compound, such as oxones and sulphones (34), also derive from the fenthion (35) type of systemic OP warblecide which was widely used in the UK prior to 1985 (2). Such metabolites, common to both fenthion and phosmet degradation, should therefore not be excluded from the list of possible candidate triggers responsible for initiating the misfolded prion. If this were the case, then fenthion, as well as phosmet, would have to be considered as a possible prion initiator.

This theory proposes that, once the offending chemical penetrates the phospholipid membranes of the CNS and attains an optimum critical concentration - as occurs in the UK-specific context of high-dose phosmet treatment - then the transformation of PrPc to PrPbse may be initiated in those cattle carrying TSE-susceptible variants of PrP and inherited deficiencies of liver detoxification enzymes.

Assuming that the OP fenthion (applied at a 5 mg/kg dose) can also play a primary role in the initiation of BSE as well as phosmet, then, because of fenthion's four-fold lesser dose rate in relation to phosmet, it would seem imperative that both direct application of systemic fenthion as well as the indirect bioconcentration route of exposure (via the recycling back of fenthion/phosmet-contaminated animal fat) must coexist before the optimum toxic concentration necessary for PrPbse initiation can be attained.

Like phosmet, fenthion also has a high solubility in lipids (9). It has been experimentally demonstrated that fenthion can bioconcentrate up to 62 times following a single step up the trophic layers of the food pyramid (36). When fenthion-treated tadpoles were fed to untreated amphibians, the amphibians bioconcentrated fenthion up to a 62-fold greater concentration than the tadpoles.

Systemic fenthion was generally used as a 'spoton' treatment in BSE-free countries like Germany, Denmark, Canada and Spain (33), where it was licensed for use on cattle. An overall lower total dosage of fenthion is applied per treatment per cow when the compound is formulated as a spot-on' treatment as opposed to when it is applied as a 'pour-on' treatment.

The UK and Eire warble-fly programmes employed pour-on fenthion extensively. The French compulsory warble-fly programme also employs fenthion as one of three systemic pour-on formulations approved as warblecides (33). Interestingly, France appears to be the only country outside of the UK whose spatiotemporal distribution of compulsory warble-fly zones bears some correlation with the spatiotemporal dynamics of their BSE incidence.

Compulsory warble eradication measures were intro-duced into France 8 years ago (33), but compulsory treatment was exclusively confined to the Brittany region at that time. This implies that the MBM derived from cattle slaughtered in the Brittany region would have passed through local slaughter houses and continuous-flow rendering plants for recycling back into cattle via concentrates produced by the local feedmills. This whole operation, coupled to importation of phosmet-contaminated MBM and live cattle from the UK into this region (as well as the recycling back of phosmet-treated pigs from the intensive units of the C6te d'Amour (see following section on bioconcentration)), would have opened up possibilities for a small degree of fenthion/phosmet bioconcentration (relative to the UK) to occur within the bovine food chain of the Brittany region, sufficient to have triggered off the small incidence of BSE (20 cases) in local cattle.

Warble treatment zones have been extended considerably over the last 3 years to cover the more southerly provinces of France.

Interestingly, six fresh cases of BSE have subsequently surfaced in these southerly regions, suggesting that the spatiotemporal distribution of BSE in France may be following warble treatment dynamics likewise, where, much like the UK, an average 4 year delayed lag exists between direct/7 indirect exposure to OP warblecide and the emer, gence of clinical BSE.

Bioconcentration of fat-bound phosmet in the farm animal food chain (see Fig. 1)

Deficiency of published research

There is little research in the literature which focuses on the bioconcentration of 0Ps in the food chain. Conclusions have been drawn from the limited amount of research that has been executed in this area (36-39), which suggests that potentially significant toxicological complications could arise in certain specific environmental contexts of OP application. Surprisingly, the most likely areas where bioconcentration of 0Ps could be presenting a health hazard, such as in the context of systemic OP warblecides, do not appear to have been investigated.

No published research exists on the bioconcentration of systemic phosmet or other warblecides within the farm animal food chain, despite their widespread intensive use in the UK farming system.

However, two studies (40) have investigated the concentration of phosmet in various cattle tissues following 'spray on' application of an aqueous based 'non-systemic' formulation of phosmet containing a low-dose, 25% concentration of active ingredient (a.i.). Although residues of phosmet in the various fat samples analysed a day after treatment did not exceed 1 p.p.m. on any count, this trial failed to screen for the highly toxic oxone and other intermediate metabolites, and also failed to acknowledge and assess existence of 'undetectable' phosmet residues that would have coupled up with phospholipid fractions.

Another study (41) investigated the problems encountered with the accumulation of certain types of OP residues (the substituted aryl derivatives) in the meat of domestic animals after application or intake of OPs, and its findings tend to contradict the findings of the aforementioned studies (40) by suggesting that a problem of OP bioaccumulation could exist in certain contexts.

But all of these studies entirely fail to reflect the specific 'in vivo' potential for bioconcentration of the high-dose systemic pour-on formulations of phosmet in the phospholipids of CNS membranes following direct application along the spinal column.

Other studies provided by the pesticide manufacturers for the World Health Organization's pesticide reviews have looked at the distribution of phosmet following oral administration of the compound (42). But the 'oral' route of phosmet entry as opposed to the systemic 'backline' route provides a much greater opportunity for hydrolytic and other degrading enzymes (abundant in the gastro tract) to metabolize the chemical before it reaches the safe haven of its lipid depots, thus preventing the possibility of any significant amount of phosmet concentrating in lipids and reaching levels of contan-fination that are toxicologically significant.

The criteria surrounding field application of systemic phosmet in the UK satisfies Hassall's four postulates for bioconcentration

Much like the organochlorine DDT, both systemic phosmet and fenthion satisfy all four of 'Hassall's Postulates' necessary for bioconcentration of chemical pollutants in the lipid phase of the food chain. Two of the postulates (43), low solubility in water and high solubility in fat, are strongly fulfilled by phosmet and fenthion (6,34,35), implying that both phosmet and fenthion possess partition coefficients (6) that strongly favour bioaccumulation in lipids.

In fact, one study (6) demonstrates that phosmet has a partition coefficient of 677, whilst other common 0Ps such as trichlorphon, dimethoate and dichlorvos have much lower partition coefficients of 3.7, 0.51 and 29, respectively. NB, trichlorphon is used as an a.i. of some warblecide brands.

Fig. 1 Bioconcentration of systemic phosmet up the farm animal food pyramid due to the practice of recycling phosmetcontarmnated bovine/porcine fat via meat and bone meal which has been manufactured by the 'continuous flow' system of rendering.

One toxicological assessment test (44) looking at various OP compounds found that a precise correlation existed between the partition coefficients and the bioaccumulation abilities of the OPs studied, hence indicating that phosmet's high partition coefficient signals a strong potential for bioaccumulation. Another assessment test (45) correlated partition coefficients with the degree of chronic toxicity that an OP exerts.

Thirdly, (43 [p. 1211), the systemic route of phosmet entry as prescribed to be 'poured along the spine', guarantees a rapid penetration and binding of lipophilic phosmet into the phospholipids of the CNS. For the systemic route of entry largely enables the chemical to bypass the batteries of hydrolytic and oxidative enzymes that are abundant in the gastro tract/liver and normally responsible for catalysing the primary pathways of phosmet degradation.

Several of the solvents (46 [p. 271) that have been more recently employed in compiling systemic formulations of OP not only guarantee rapid penetration of phosmet into lipid depots, but the actual toxicological properties of the solvent itself may also impair hydrolytic enzyme activity, thus enabling phosmet to evade enzymic degradation whilst in passage to the relative safety of its lipid depots.

Once phosmet, like other lipophilic OPs, is bound into the lipids and non-vital sites (46 [p. 41, p. 481) (47), hydrolases are prevented from accessing the P-O bonds of the phosmet molecule for catalytic attack and primary degradation. Hence, phosmet becomes locked into the lipid, which acts like a sort of molecular 'lobster pot' (43) until times of stress cause a sudden surge of demand for fat assimilation, with the subsequent decoupling and release of phosmet into the general circulation. Other lipophilic 0Ps such as leptophos (48) and fenitrothion (47) have been shown to bind and decouple with mammalian fat in the 'in vivo' context in this way.

And fourthly (43), if phosmet bioconcentration is to be fulfilled up through the food pyramid, then the predator at each trophic level (e.g. the cannibalistic cow in this context) needs to ingest a constant supply of phosmet-contaminated fat at a rate that is sufficiently speedy to outmatch the body's rate of degradation and disposal of the chemicals.

Adult UK dairy cattle ingested significant quantities of animal fat on a daily basis. For animal fat became a significant constituent of MBM-containing concentrates once solvent extraction was phased out of the UK rendering process in the early 1980s (49) and was fed up until the MBM ban in 1988, and the feeding of animal tallow to cattle in artificial milk powders, etc. was not prohibited until 199 .

John Wilesmith's research (50) postulates that BSE will erupt in those adult cows who were fed brands of concentrates as young calves which contained particularly high percentages of MBM protein. However, as this MBM was processed via the continuous-flow rendering system, which did not employ solvent extraction, such feed would have retained the tallow fat fraction that carries any fat-bound phosmet contaminants that may be present. This MBM therefore presents a more severe threat of toxicological hazard to young calves than if fed to adult cattle. For, much like all species of mammalian infant, young calves possess immature enzymic detoxification systems (51) that are incapable of efficient detoxification of chemical pollutants that enter their body systems. An even greater degree of 'cliemical susceptibility' would apply to the context of the calf fetus that has been exposed to phosmet via a placental intake of phosmet-contaminated lipid; originating either from a source of direct phosmet treatment upon the mother cow or via ingestion of phosmet-contaminated MBM concentrates by the mother cow. For phosmet has been demonstrated to cross the placenta (34,52) and, following a 20 mg/kg application onto pregnant rats (52), has been shown to concentrate at nearly two-fold greater concentration of 380 ng/g in the liver of the fetus as opposed to the 215 ng/g concentration found in the liver of the treated mother itself. These findings add strong support to this theory whose central tenet proposes 'in utero' exposure to phosmet as the most likely developmental stage for initiation of the BSE prion.

Studies have demonstrated teratogenic complications such as hydrocephalus (53) following exposure to 1.5 mg/kg oral daily doses of phosmet during the critical 'window' period of gestation. Another lab formulation of an organo-phthalimido-phosphorus (similar to phosmet) has produced severe teratogenic complications at low doses (54), whilst high doses of the warblecide 'trichlorphon' used upon pregnant pigs between 55 and 70 days post conception has produced hyperplasia of the cerebellum accompanied by severe ataxia and tremors in the offspring piglets (55).

Considering all four of 'Hassall's postulates' for bioconcentration have been satisfied in relation to exposure of the UK bovine to systemic phosmet during the 1980s, then, despite the absence of any direct analytical evidence relating to this phenomenon, it would still seem reasonable to assume that bioconcentration probably occurred in the UK context of compulsory high-dose systemic usage.

Experimental evidence fo bioconcentration of organophosphorus compounds

Two separate trials involved feeding kestrels (37) and amphibians (36) upon frogs and tadpoles maintained in water that was deliberately contaminated with a range of doses of the 0Ps parathion, malathion, acephate and fenthion. Both trials demonstrated that significant degrees of bioconcentration can take place with 0Ps in specific 'in vivo' contexts.

In one of the trials (36), the untreated amphibians bioconcentrated fenthion and parathion from treated tadpoles to an average order of 62 and 64 times respectively.

A report (38) from the wild demonstrates how predatory birds, such as barn owls, eating prey poisoned by the OP warblecide famphur, bioconcentrated the chemical to the extent that they developed secondary poisoning. Another report (39) demonstrates how the widespread deaths of Swanson's hawks as a result of monocrotophos poisoning in Argentina may have resulted from secondary poisoning after the hawks had ingested grasshoppers that were contaminated with this OP after its application onto lucerne.

Both of these field incidents and 'in vivo' trials thus demonstrate how sufficient quantities of lipophilic 0Ps can pass through the digestive tract without undergoing hydrolysis and degradation. Once 0Ps have successfully reached the storage fats, significant degrees of bioconcentration can take place that could have catastrophic toxicological consequences in certain contexts.

Toxic impurities can exacerbate the bioconcentration factor

It should also be noted that common impurities such as OOS-trimethyl phosphorodithioate (42,56) and N-chloromethylphthalimide (42,57) that are found in association with technical formulations of systemic phosmet (the former is found in fenthion also) will actually inhibit crucial hydrolases (46 [p. 83]) such as the A esterase, carboxylesterase, providing the impurity is present at a sufficiently high percentage proportion of the compound. This would have the overall negative effect of preventing degradation of the OP compound itself. Some specific types of 'neuropathic' OP parent compound also inhibit hydrolases (58), thus inactivating their own degradation with disastrous consequences for the individual contaminated with such a compound.

Bioconcentration of low levels of systemic phosmet used upon pigs as a part causalfactorfor the low incidences of endemic BSE in Switzerland and France

In Switzerland and France, countries suffering a relatively low incidence of endemic BSE, systemic phosmet is only licensed for use upon pigs. Because of the lipophilic nature of systemic phosmet, the chemical must theoretically bind into the fat of the treated pigs and then further bioconcentrate into the fat of any animals ingesting that contaminated fat on a regular basis.

Pig fat is included in virtually all brands of cattle concentrated feeds in Switzerland, incorporated at rates of 1.5%-10% of the total ingredients (Personal communication; Dr D Guidon, Swiss Federal Research Station for Animal Production, CH-1725 Posieux, Switzerland). Various forms of straight and protected fat, etc. are also fed.

In France, 20 out of the 26 cases of reported BSE have occurred within the C6te d'Amour region of Brittany, which is also home to the most intensive centre of pig production in Europe. Systemic phosmet was intensively used on the pigs here, and the rendered pig offal was recycled back into the dairy herds of this same vicinity via the MBM ingredient that was milled up into concentrates by the local animal feed compounders. The relatively high incidence of BSE in this locality may be explained by the use of phosmet in these intensive pig units, and the consequent potential for its bioconcentration in cattle. NB, pigs do not develop TSE after exposure to phosmet themselves because they are treated at the 2 mg/kg low-dose rate, whilst pigs, as a species, do not carry the TSE susceptible types of PrP genotype.

All countries affected with endemic BSE have subjected their cattle and/or pigs to a combined exposure of both direct systemic phosmet treatment as well as indirectly to the bioconcentrated residues of phosmet via feeds containing animal fat. Such fat has to be sourced from rendered-down livestock that have originated from any country (e.g. UK), where treatment with systemic phosmet has been intensive. The intensity of phosmet exposure within each country will be proportional to that country's incidence rate of endemic BSE.

MBM produced after cessation of solvent extraction favours the retention of the tallowfraction and its phosmet-bound contaminants in thefeed

Analogous to the infamous DDT debacle, countries which employed both systemic phosmet treatments and continuous-flow MBM production where phosmet-contaminated fat was continually recycled, opened themselves up to a vicious circle of phosmet bioconcentration up through the farm animal food pyramid. The UK's 160 000-head BSE epidemic can be explained by their most intensive reliance upon these prerequisites.

The potential UK problem of exposing cattle to OP contaminants in tallow was activated further once solvent extraction was phased out in the MBM plants in the early 1980s (49), making way for the more lucrative 'continuous-flow' system of rendering. This cessation of solvent extraction permitted the tallow fat fraction, which contained the fat soluble phosmet contaminants derived from rendered-down pigs and cattle, to remain as a stable component of MBM. Once bound into the fat depots, both phosmet parent compound and some of its toxic metabolites are capable of evading the usual degradation processes of hydrolysis, etc. encountered in the actual rendering process of MBM as well as in the digestive tract of the animal ingesting that feed.

In places like Guernsey island where there was a high incidence of BSE (8) with a relatively small amount of direct systemic phosmet usage (applied for the voluntary control of cattle lice and mange as a 10 mg/kg treatment with a follow-up dose 14 days later), there was an intensive 'satellite' dependence upon 'continuous-flow' MBM imported from the UK. Furthermore, a high proportion of fat was required in concentrate feeds used on Guernsey to cater for the production of high-fat Channel Island milk which has to be intensively produced on the island (e.g. demanding a high input of concentrated feeds) due to the competitive demand for a limited amount of high-priced agricultural land.

Importation of UK-rendered MBM into countries suffering low levels of endemic BSE

It should also be considered how the importation of UK manufactured 'continuous flow' MBM into countries affected by low incidences of endemic BSE, not only conveyed a source of bioconcentrated phosmet but also assisted in the spread of low levels of the hypothetical phosmet-modified PrPbse. This could have been partly responsible for causing the relatively much lower incidences of BSE in these countries.

Providing that this exogenous source of PrPbse is able to resist trypsin degradation in the gastro tract and cross the gut and blood-brain barriers as an intact 'infectious' prion, then it seems possible that BSE can be reproduced within any individual recipient of these recycled prions providing their PrP genotype is predisposed to this specific new variant TSE disease.

Table Correlating criteria of phosmet usage with incidence rates of endemic BSE in various countries (includes all phosmet-using countries)

It should also be noted that cattle which were phosmet treated in the UK and later imported into countries such as Portugal and France, where they were eventually slaughtered, would have also lead to small amounts of phosmet-modified PrP/phosmetcontaminated fat entering the MBM food chain of the home reared cattle in these countries.

As can be seen from the table of data covering all of the countries that employ phosmet - as well as some of the countries that do not - the intensity of usage of systemic phosmet within each country predicts, with a fairly high degree of reliability, the incidence rate of endemic BSE within that country.

Proposed mechanisms for a phosmet-induced abnormal post-translational covalent modification of the prion protein as initiator of BSE pathogenesis

In vitro experimental challenge

Preliminary results of an 'in-vitro' experimental challenge which exposed tissue culture cells expressing PrP to low doses of phosmet support this hypothesis.

Dr Stephen Whatley of the Department of Neuroscience, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK, challenged PrP tissue culture cells with 2 p.p.m. and 12 p.p.m. doses of phosmet. Phosmet interacted dramatically with PrP, causing PrP to traffic and distribute abnormally in the cells. In the phosmet-treated cells, three out of the four hitherto recognized abnormal characteristics (7) specific to the TSE causing pathogenic PrP isoform were invoked. The alterations involved an increase in the expression of PrP in surface cells, an accumulation of PrP in the microsomes (lysosomes and golgi, etc.) and an abnormal resistance of the membrane anchored PrP to cleavage by phospholipase C. These phosmet-induced reactions were specific to the prion protein and were proportional to the dose of phosmet introduced. This model failed to detect protease resistance in phosmet-affected PrP.

The increase of PrP at the lysosomes as well as the abnormal resistance of membrane bound PrP to phospholipase C cleavage suggests that some abnormal PrP modification has been invoked by the introduction of phosmet. Interestingly, 'in-vitro' trials with brain cells that have been affected by an abnormal scrapie PrP (PrPsc) isoform (7,8 [p. 571) demonstrates a similar abnormal pattern of PrP distribution and PrP phospholipase C resistance as invoked in these trials with phosmet. PrPsc diseased cells also demonstrate the same increase in cellular expression of PrPc (7,8 [p. 57]) as demonstrated in the phosmet-PrP trials.

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)

stores of calcium

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.

Organophosphorus compounds interact with phosphatidylinositols on sperm membranes

Interestingly, PIS also play a major role in membranemediated processes of mammalian fertilization such as sperirn capacitation and the acrosome reaction in the epididymis (75). Research into the negative effects of low-level OP exposure on male fertility has demonstrated that 0Ps bind to specific membrane receptors on sperm, affecting their motility via the 'knock on' effects of OP's increasing protein kinase C turnover and abnormally phosphorylating the Pls on sperm membranes (75). Thus it could be speculated that once a high enough concentration of OP has bound onto any sperm cells that ultimately succeed in successful fertilization, those OP-modified. PIS will therefore 'infect' the phosphoinositide system of the fertilized embryo with a predisposition to phosphorylate PIs at an abnormal site, thus transmitting disease onto the next generation in a novel manner which does not implicate a mutagenic effect.

Perhaps such a hypothetical mechanism may also clarify the transmissible element of Gulf War Syndrome, where Gulf War veterans seem able to transn-fit the disease onto the ' ir wives/embryos, putatively through their sperm, as well as explain the many anecdotal stories circulating the farming grape-vine of BSE transmitting through bull semen derived from BSE-suffering bulls; notably the story of the bull which was exported (necessitating treatment with OP warblecide) to a herd in N. Ireland where he subsequently developed BSE, only to find that many of his daughters who were reared up into that herd subsequently contracted BSE.

The same mechanism of transmission of phosmetmodified PIs could account for the vertical transmission of BSE from mother to calf.

Much like 0Ps, lithium has also been shown to disturb the brain phosphoinositide turnover (78,82), although it has been postulated to disrupt the cycle at a different point from 0Ps, by competing with magnesium ions that are essential for the binding of GTP to G proteins and the subsequent activation of phospholipase C. Arriving at the same net effect via a different avenue, it could be postulated that, once the organism is chronically exposed to low doses of OP, turnover of the phosphoinositide cycle is agonized to such an elevation, that cells are eventually depleted of available magnesium (as occurs following prolonged convulsions) so that the cycle has to collapse.

Interestingly, treatment of early-stage BSE-suffering bovines with magnesium sulphate has produced longterm remission of symptoms (2).

It is of note that the literature cites a few cases of a reversible CJ1)-like syndrome in psychiatric patients, which has been attributed to the long-term chronic effects of prolonged lithium therapy, presumably only erupting in susceptable genotypes (83).

Mechanism 2: Phosmet-induced phosphorylation of a phosphotyrosine residue on the prion protein as an 'in-utero' early-life modification which initiates the TSE disease process; expansion of the concept already proposed

All classes of OP compound are known to phosphorylate and modify the serine active site on serine esterase/protease groups of enzymes such as acetylcholinesterase, but certain multi-site binding types of OP such as phosmet can act as an exogenous phosphorylating agent by interacting with several endogenous, non-enzymic phosphorylation sites (e.g. tyrosine, histidine, arginine, etc.) on membrane proteins (84). This ultimately disrupts the conformational structure of these proteins so that they can no longer perform their correct metabolic activity.

Protein tyrosine phosphatases and tyrosine kinases play a crucial role in influencing the activity of various cell membrane receptors such as the 'integrins', which reinforces the well-recognized role that tyrosine phosphorylation plays in cell adhesion (85).

It has been demonstrated that some types of phthalimide compound upregulate endogenous phosphorylation of tyrosine residues on many membrane proteins (86), including the various 'integrin' receptors (87,88), thus suggesting that phthalimides can exert an exogenous influence upon cell adhesion.

Recent toxicological studies with various phthalimide compounds, such as the phthalidomide compound, has demonstrated a marked loss of density of integrin adhesion receptors in the various fetal cells after treatment with phthalimide, with a corresponding alteration in expression of cell-cell, cellextracellular matrix interactions occurring as a result (87,88). These alterations in cell adhesion molecules are suggested to be the long-sought primary mechanism of teratogenic action invoked by some of the phthalimide family of compounds. Such an effect upon cell adhesion explains why the phthalidomide drug is employed to block intercellular signalling that, in turn, blocks the autoimmune mediated rejection mounted in host-versus-graft transplants.

There is a varied mode and degree of disruption of the cell adhesion system depending upon the type of phthalimide compound employed (87,88). It is proposed that 'in-utero' exposure to the Nmercaptomethyl phthalimido moiety of phosmet will cause a subtle degree of disturbance to the cell adhesion system by altering the conformational shape of the 'prion protein' adhesion molecule - prion protein having been postulated to serve as an adhesion molecule (89) - which, although not causing any immediate acute clinical disturbance, leads on to prion disease years later; as opposed to the full-scale down-regulation of adhesion receptors on limb bud cells, etc. with severe teratogenic consequences that are invoked by the phthalidomide type of phthalimide (88).

It is proposed that phosmet interaction with PrP causes an abnormal phosphorylation on a tyrosine residue of PrPc, perhaps due to the phosmet-induced generation of free radicals that are free to pair up with and disrupt essential free radicals carried in the tyrosine residues of PrP by blocking out normal electron donors from other active sites. This leads to a conformational change which alters the cell-cell, cell-extracellular matrix interactions of PrP, thus blocking various signal pathways that are crucially important in the immune response, etc. The resulting abnormal PrP isoform is protease-resistant.

It should also be noted that phosmet crosses the placenta (34,52) concentrating in the fetus at nearly twice the concentration as found in the treated mother (52). Phosmet also produces teratogenic defects, such as hydrocephalus, in offspring whose mothers were exposed to oral daily doses of 1.5 mg/kg phosmet around day 13 of gestation (34,53).

In search of aetiological triggers underlying the new variant and sporadic CJDs, it would seem pertinent to look at exposures to various 'abusive' hallucinogenic indoles, tricyclic anti-depressants and OP-based products (head-lice shampoos, pet-flea shairnpoos/collars, OP flame retardants - as used in blankets and by firefighters, etc.) and other types of pesticides that act like phosmet, cuprizone and disulfiram (SE-inducing chemicals) via inhibition of monamine oxidase (MAO) (30,34,51 [p. 2701). etc., as serotonin agonists in common, and upregulate phosphorylation of phosphotyrosine residues on membrane proteins in susceptible genotypes.

Mechanism 3: Phosmet's sulfonyl or phthalimido metabolite induces a subtle alkylation/acylation of a target site at some stage of PrP translation synthesis

A: Phosmet inhibits lysosomal glycosidases leading to the development of a hyperglycosylated PrP isoform which has failed to undergo deglycosylation. 0Ps that express alkylating activities have been shown to inhibit lysosomal enzyme secretion such as lysozyme and glucuronidase (13). Systemic poisoning by an OP with subtle alkylating activity, such as phosmet (6), could account for the intensively glycosylated pattern of PrP that has been identified as a hallmark of the BSE and new-variant CJD prion (1). The OP inhibits a PrP specific glycosidase, which prevents the deglycosylation of the glycosylated bands, producing a hyperglycosylated PrP that subsequently disrupts the correct conformational assembly of itself.

Differing patterns of PrP glycosylation have been reported to differentiate the 'strains' of abnormal PrP isoform that characterize the CJD variants (1). But the new-strain CJD and BSE PrP isoforms exhibit the same distinct intensive glycoform pattern, perhaps resulting from an abnormal uniform type of post-translational N-linked glycosylation modification. On the other hand, the intensively glycosylated bands on PrP could simply demonstrate the fact that relatively younger mammals (including young BSE cattle and adolescent CJD cases) will naturally glycosylate their PrP and other glycoproteins more intensively than more elderly mammals (including familial/sporadic CD cases).

It has already been suggested that subclinical, chronic poisoning of sheep with naturally occurring plant alkaloids could initiate scrapie by inhibiting glycosidases within cells that express PrP (14). This leads to a build-up of glycosylated PrP which is prevented from deglycosylation. The primary structure of PrP is altered following the inhibition of glycosidases, for glycosidases would normally lyse the esters that link glycan chains onto other molecules.

B. Phosmet intoxication of the cell invokes a subtle reversible covalent modification/mutation at the PrP DNA or PrP mRNA translational stages of PrP synthesis. Phosmet has been shown to exert subtle mutagenic disturbances in several 'in-vivo' and 'invitro' contexts (34,90-95). The sulphonyl metabolite of phosmet could exert mild alkylating effects on the sulfhydryl group of methionine or cysteine, the imidazole nitrogen group of histidine, or on the nitrogen group of guanine, etc. of target proteins or DNA itself (51 [p. 687]). But such a mechanism seems unlikely, given that it is hard to demonstrate factors which explain why phosmet should confine its alkylating attack to the genetic material of PrP. And, more particularly, the amino acid sequence of normal cellular bovine PrP duplicates the same sequence found in PrPbse, suggesting that the aetiological trigger is confined to some abnormal post-translational modification of PrP.

But it cannot be ruled out that some hitherto unrecognized chemically induced mutation of a PrP specific chaperone, isomerase, ubiquitin, or p53 gene which could permit the generation and/or survival of a misfolded PrP isoform, is more likely to occur when the organism is subjected to a stress challenge which results in the increased turnover of PrP synthesis and folding.

The phthalimide moiety of phosmet, known to interfere with DNA and RNA translation (34,95,96), could be exerting some subtle, reversible disturbance at the RNA mid-translational stages of PrP synthesis, such as a temporary reversible frame shift type mutation which can be invoked by a wide variety of low-dose chemical exposures (51 [pp. 639-648]).

Interestingly, various types of phthalimide, rather than targetting DNA/RNA in a direct covalent manner, have been demonstrated to significantly alter the activities of a whole range of enzymes such as polymerases and synthases which are integrally involved in controlling the rate of protein synthesis (96).

There is one way in which phosmet could actually invoke a mutation by deleting or sticking/binding to select PrP RNA codons which would lead to the synthesis of an abnormally folded PrP that still retains the same amino acid sequence as the normal PrP isoform.

Select regions of 'unfavoured' codons are directly involved with controlling RNA translational pauses (97) which are crucial for the balanced folding of protein. The phthalimide or other moiety of phosmet could disrupt the normal 'codon bias' flow of PrP translation by knocking out this region of unfavoured codons - regions which are essential for promoting balanced folding of proteins due to temporally separating folding of the specific regions of their polypeptide chains/structural domains (97).

Site-directed mutagenesis has been used in trials (97) to remove this region of translational pause from the TRP3 gene in yeast cells, resulting in incorrect intracellular folding of the enzyme in its secondary/ tertiary stages; whereby a misfolded isoform, predominated by excessive beta sheet structure formation, is produced - due to a change in mRNA secondary structure formation. Such an abnormal conformation produced here equates to the model of the conformationally deranged prion protein isoform proposed by Stanley Prusiner, that is heralded as the cause of prion degenerative disorders.

Organophosphorus-induced susceptibility of gastro and blood-brain barrier towards passage of recycled PrPbse contained in feeds derived from bovine CNS tissues

0Ps are widely, recognized to inhibit serine esterases such as trypsin and chymotrypsin. As sporadic and conventional prion variants are supposedly degraded by trypsin in the healthy and intact gastro tract, oral intake of significant doses of OP residues in feed could theoretically invoke susceptibility to prion intake across the gut wall because of their direct inhibition of trypsin activity (98) as well as disrupting permeability gradients across the wall (99) or affecting neuroendocrine mediation of 'Peyer's patches' in some way. Or perhaps the new variant prion is conformationally modified in a manner that blocks trypsin from accessing its normal cleavage/degradation sites.

Thus an OP such as phosmet, may not only be initiating the endogenous production of the prion agent within the bovine CNS, but also enabling an exogenous source of recycled prions (from rendereddown bovine CNS tissues in meat and bone) to cross the gut wall and blood-brain barrier (100,101), and thus re-enter the CNS - hence, initiating and exacerbating the bioaccumulating 'prion pyramid' effect.

0Ps could also be compromising the bovine's susceptibility to prion disease by altering the metabolism and permeability gradients of CNS neurones and their plasma membranes, thus facilitating transmission of exogenous PrPbse back into the cell where fresh supplies of normal PrPc are available for conversion to PrPbse.

Other factors such as ulceration of the gastro tract due to chronic daily intake of chemical silage additive residues (based on formic acid) for instance, or gastrointestinal infestations of parasitic nematode worms (102) could create sufficient physical damage to the gut membranes to permit leakage of prions into the internal body environment.

Similarly in relation to human new-variant CJD; ulceration, gut worms, gastro surgery or exposure to pesticidal or 'abusive' recreational psychotrophic drugs which agonize serotonergic/peptide turnover in the gut and CNS (103), could all lead to the physical or biochemical impairment of gastro and blood-brain barrier permeability gradients which might normally be protecting the human against intake of prions from contaminated feed.

Initiation of pathogenic PrP in new-variant CJD.

The UK government's Spongiform Encephalopathy Advisory Committee (SEAC) have consistently pointed out that people occupationally involved with pets or farm animals are more at risk of developing 01) (8). As the licensing of phosmet in the UK has been exclusively limited for use upon farm animals and pets, then it would seem relevant to consider phosmet as a possible common cause of nvCJD as well as a common cause of BSE in cattle. Chemical exposures can be hypothetically correlated to the clusters of CJD that have emerged.

Dr Randall Crom of the Epidemic Intelligence service of the Arizona Department of Health Services at Phoenix reports that a cluster of six cases of sporadic/famililial CJD variants has emerged around a missile production plant in Tucson, Arizona from between 1980 and 1986. MAO inhibiting chemicals (e.g., serotonin agonists) such as cuprizone are invariably employed as propellants in the manufacture of missiles. Cuprizone induces a type of spongiform encephalopathy (30). Three of these CJD cases implicated workers occupationally involved at this plant who were diagnosed between 1985 and 1986.

Another cluster of six confirmed/suspected CJD cases to date is currently emerging in people who have worked or resided in villages 10 miles east/south of an OP insecticide manufacturing plant sited at Yalding in Kent. On 17 April 1986, there was a major leak of an OP gas from the plant which had an 'offensive' odour (Kent Messenger 18 April 1986). The 10-year delay prior to manifestation of C.11) symptoms in susceptible residents averaging 10 miles downwind of this plant could be postulated as the incubation period of the CID.

The fact that this CJD cluster to date has largely only implicated individuals who were occupationally and residentially confined to the rural areas of the 'Weald' district of Kent, could also be part explained by the unique presence of hop and fruit farms within this region of the UK which utilize a 100-fold greater intensity of systemic OP applications per acre in 1983 than were applied upon the common types of cereal/ arable crops grown all over the UK (104).

The villages where these CJD victims resided or were temporarily employed - High Halden, Mersham, Bethersden, Sissinghurst, East Peckham- were all home to hop farms that applied a range of the highly toxic 'class 2' types of systemic 0Ps containing sulphur such as TEPP, omethoate, mephosfolan, methidathion and oxydemeton-methyl, which, on the greater part, contain the same 0,0-dimethyl phosphorodithioate moiety as found in phosmet (40).

Those who were occupationally involved or living near to these hop fields in the sheltered valleys of the Weald would invariably be chronically exposed to the atmospheric 'fall-out' of low doses of these 0Ps after their field application.

Acknowledgement

1 express my gratitude to the Murray Trust for their grant support for costs incurred in studying BSE epidemiology/OP usage on farms in Switzerland and France.

References

1. Collinge J, Sidle KC L, Meads J, Ironside J, Hill A F. Molecular analysis of prion strain variation and the aetiology of 'new variant' CID. Nature 1996; 383: 685-690.

2. Purdey M. The UK epidemic of BSE: Slow virus or chronic pesticide initiated modification of the prion protein. (Pts 1 and 2). Med Hypotheses 1996; 46: 429-454.

3. Scharff D M, Sharman G A M, Ludmig P. Illness and death induced by treatments with systemic insecticides for the control of cattle grubs. J Am Vet Med Ass 1962; 141(5): 582-587.

4. Andrews A H. Abnormal reactions and their frequency in cattle following the use of OP warble fly dressings. Vet Record 1981; 109: 171-175.

5. Sergeev GB, Sergeev BM, Nekrasova G N. Influence of phthalophos on water permeability of artificial lipid membranes. Biofizika 1982; 27(3): 548-550.

6. Dedek W, Grahl R, Schmidt RA. A comparative study of guanine N-alkylation in mice in vive, by the OP insecticides trichlorphon, dimethoate, phosmet and bromophos, Acta Pharmacol Toxicol 1984; 53: 104-109.

7.Gabizon R, Prusiner S. Prion liposomes. Biochem J 1990; 266: 1-14.
8.SEAC. TSEs: A summary of present knowledge and research. HMSO, 1994.

9. Good J L, Khurana R K, Mayer R F, Cintra W M, Albuquerque E X. Pathophysiological studies of neuromuscular function in subacute OP poisoning induced by phosmet. J Neurol Neurosurg Psychiatry 1993; 56: 290-294.

10.Johnson M K. Contemporary issues in toxicology. 0Ps and delayed neuropathy. Toxicol Appl Pharmacol 1990;102(3): 385-399.
11.Dauterman W C. Biological and non biological modifications of OP compounds. Bull WHO 197 1; 44: 133-150.

12. Katz L S, Marquis J K. OP induced alterations in muscarinic receptor binding and phosphoinositide hydrolysis in the human SK-N-SH cell line. Neurotoxicology 1992; 13: 365-378.

13. Becker E L, Koza E P, Sigman M. OP inhibition of lysosomal enzyme secretion from polymorphonuclear leucocytes. Immunology 1978; 35: 373-380.

14.Dealler S. Alkaloidal glycosidase inhibitors as the cause of sporadic scrapie. Med Hypothese 1994; 42: 69-75.

15. Johnson P S, Michaelis E K. OP interactions at the NNIDA receptors in brain synaptic membrane. Mol Pharmacol 1992; 41(4): 750-760.

16.Snyder S H, Bredt D S. Biological roles of nitric oxide. Sci Am 1992; May: 28-35.

17. Matkovics B, Szabo L, Mindszenty L, Ivan J. The effects of OP pesticides on some liver enzymes and lipid peroxidation. Gen Pharmac 1980; ll: 353-355.

18. Stahl N, Baldwin M A, Teplow D B et al. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 1993; 32: 1991-2002.

 19. EI-Fawal H AN, Correll L, Gay L, Ehrich M. Protease activity in brain, nerve and muscle of hens given neuropathyinducing 0Ps and a calcium channel blocker. Toxicol Appl Pharmacol 1990; 103: 133-142.

20. Scifert J, Casida J E. Possible role of micrombules and associated proteases in OP induced delayed neuropathy. Biochern Pharmacol 19U; 31(1l): 2065-2070.

21. Katoh K. A review of the studies of the delayed neurotoxicity induced by OP esters. Sangyo-Eiseigaku-Zasshi 1995; 37(5): 309-319.

22.Lotti M. The pathogenesis of OP polyneuropathy. Crit Rev Toxicol 1992; 21(6): 470-486.

23. Cashman N R, Loertscher R, Nalbantoglu J et al. Cellular isoform of the scrapie agent protein participates in lymphocyte activation. Cell 1990; 61: 185-192.

24. Katsenovich L A, Ruzybakicv R M, Fedorina L A. The T and B systems of immunity in pesticide intoxication sufferers. Gig Tr Prof Zabol 1981; 4: 17-19.

25. Casale G P, Cohen S D, Dicaptia RA. The effects of OP induced stimulation on the antibody response to sheep erythrocytes in inbred mice. Toxicol Appl Pharmacol 1983; 68:198-205.

26. Telling G C, Scott N, Mastrianni J et al. Prion protein propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 1995; 83:79-90.

27. Lasmezas C 1, Deslys J-P, Robain 0 et al. Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science 1997; 275: 402-403.

28. Whatley SA, Powell J F, Politopoulou G, Campbell 1 C, Brammer M J, Percy N S. Regulation of intracellular free calcium levels by the cellular prion protein. Mol Neurosci 1995;6:2333-2337.

29. Kristensson K, Feuerstein B, Taraboulos A, Hyrm M, Prusiner S, DeArmond S. Scrapie prions alter receptor-mediated calcium responses in cultured cells. Neurol 1993; 43: 2335-2341.

30. Venturini G. Enzyme activities and sodium, potassium and copper concentrations in mouse brain and liver after cuprizone treatment in vivo. J Neurochern 1973; 21: 1147-1151.

31. Pictrasanta Y. Proceedings of Visioconference Nache Folle et Pesticides' 1997 April 3rd. Paris, Rennes, Montpelier, Lyon. Groupe des Ecologistes, 78 route de Paris - BP 19 - 69751 Charbonni~res-les-Bains, Cedex.

32.O'Keefe M, Eades J F, Strickland K L. Phosmet residues in milk following treatment of dairy cows for warble fly. J Sci Food Agri 1983; 34: 463-465.

33.Liebisch A. Hypoderma infestation in cattle. The spread and combatting of hypoderma infestation. of cattle in countries of the EEC. VET. Lebliard. Verlag GmbH, Lebliardsweg 2a 7750 Konstartz, Germany, 1988.
34.Phthalophos. In: Izincror N F, ed. Scientific Reviews of Soviet Literature on Toxicity and Hazards of Chemicals,
No 46: 1-21. USSR State Committee for Science and Technology/United Nations Environment Programme, 1983.
35.O'Keefe M, Eades J F, Strickland K L. Fenthion residues in milk and milk products following treatment of dairy cows for warble fly. J Sci Food Agri 1983; 34: 192-197.
36 Hall R J, Kolbe E. Bioconcentration of OP pesticides to hazardous levels by amphibians. J Toxicol Environ Health 1980;6:853-860.
37 Fleming W J, de Chacin H, Patee 0 H, Lamont T G. Parathion accumulation in cricketfrogs and its effect on American kestrels. J Toxicol Environ Health 1982; 10: 921-927.
38 Hill E F, Mendenhall V M. Secondary poisoning of barn owls with famphur, an OP pesticide. J Wildlife Mgmt 1980; 44:676-681.
39 Buffin D. Death of Swanson hawks in Argentina due to OP pesticide poisoning. Pesticide News 1997; 35(l): 6.
40 Report of the 1976 joint meeting of the FAO panel of experts on pesticide residues and the environment. WHO technical report, 1977 Series No 612. Plant Production and Protection Series No 8, pp 491-533.
41 Kaemmerer K, Buntenkotter S. The problem of residues in meat of edible domestic animals after application or intake of OP esters. Residue Review 1973; 46:1.
42 Pesticides Residues in Food (1994). Report sponsored by FAO and WHO 127:154.
43 Hassall K A. The chemistry of Pesticides. London: Maciffillan Press, 1983.
44 Freed V H, Chiou C T, Schmedding D, Kohnert R. Some physical factors in toxicological assessment tests. Environ Health Perspectives 1979; 30: 75-80.
45 Freed VH, Haque R, Schmedding D, Kohnert R. Physico-chemical properties of some 0Ps in relation to their chronic toxicity. Environ Health Perspectives 1976; 13: 77-8 1.
46 Environmental Health Criteria 63, OP insecticides; a general introduction. Geneva: WHO, 1986.
47 Ecobichon D J, Ozere R L, Reid E, Crocker J F S. Acute fenitrothion poisoning. Can Med Ass J 1977; 116: 377-379.
48 Yamauchi T. Effect of experimental thinness and obesity on the delayed neurotoxicity of the OP pesticide phosvel. Nippon Eiseigakri Zasshi 1980; 35(5): 782-793.
49 Wilesmith J W, Ryan J B M, Atkinson M J. BSE: epidemiological studies on the origin. Vet Record 1991; 128: 199-203.
50 Wilesmith J W, Ryan J B M, Hueston W D. BSE:Case control studies of calf feeding practices and meat and bone meal inclusion in proprietary concentrates. Res Vet Sci 1992; 52:325-331.

51 Goldstem A, Aronow L, Kalman S M.. Principles of Drug Action: Basis of Pharmacology, 2nd edn. Wiley International Edition. New York:John Wiley, 1974.

52 Ackermann H, Engst R. Presence of OP insecticides in the fetus. Arch Toxicol 1970; 26: 17-22.

53 Martson L V, Voronina V M. Experimental study of the effect of a series of phophoro-organic pesticides (Dipterex and Imidan) on embryogenesis. Environ Health Perspectives 74. 1976;13:121-125.

54. Robens J F. Teratogenic activity of several phthalimide de- rivatives in the golden hamster. Toxicol AppI Pharmacol 75. 1970;16:24-34.

55. Knox B, Askaa J, Basse A et al. Congenital ataxia and tremor with cerebellar hypoplasia in piglets born by sows treated with trichlorphon. Nord Vet Med 1978; 30: 538-545.

56. Toia R F, March R B, Umetsa N, Mallipudi N M, Allahyari R, Fukuto T R. Identification and toxicological evaluation of impurities in technical malathion and fenthion. J Ag Food Chem 1980;28:599-604.

57. Ackermann H, Faust H, Kagan Y S, Voronina V H. Metabolic and toxic behaviours of phthalimide derivatives in albino rat. Arch Toxicol 1978; 40: 255-261.

58. Chemnitius J M, Zech R. Inhibition of brain carboxylesterase by neurotoxic and non-nenrotoxic OP compounds. Mol Pharmacol 1983; 23: 717-723.

59. Viana G B, Davis L H, Kariffinan F C. Effects of OP and nerve growth factor on muscarinic receptor binding number in rat pheochromocytoma PC12 cells. Toxicol Appl Pharmacol 1988;93:257-266.

60. Jerkins A A, Katiftman F C. Effects of soman on neuritic outgrowth and substrate utilisation by explants of the superior cervical ganglion. Toxicol Appl Pharmacol 1984; 75: 240-245.

61. Tuler S M, Bowen J M. Toxic effects of 0Ps on nerve cell growth and ultrastructure in culture. J Toxicol Environ Health 1989;27:209-223.

62.Russell R W, Overstreet D H. Mechanisms underlying sensitivity to OP compounds. Prog Neurobiol 1987; 28: 97-129.

63. Katiffinan F G, Davis L 1I, Whittaker M. Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by OPs. Biochem Pharmacol 1990; 39(2): 347-354.

64. Gant D G, Eldefrawi ME, Eldefrawi AT. Action of OP on GABA receptors and voltage-dependent chloride channels. Fundam Appl Toxicol 1987; 4: 698-704.

65. Walenga R, Vanderhoek J Y, Feinstein M B. Serine esterase inhibitors block stimulus induced mobilization of arachidonic acid and phosphatidylinositol-specific phospholipase C activity in platelets. J Biol Chern 1980; 255(13): 6024-6027.

66. Davies D E B, Holub B J. Comparative effects of OP insecticides on the activities of acetylcholinesterase, diacy1glycerol kinase and phosphatidylinositol phosphodiesterase in rat brain microsomes. Pesticide Biochem Physiol 1983; 20: 92-99.

67. Mckinley M P, Longo F M, Valletta J S et al. Nerve growth factor induces gene expression of the prion protein and amyloid protein precursor in the developing hamster central nervous system. Progr Brain Res 1990; 86(19): 227-237.

68. Stahl N, Borchelt D R, Prusiner S B. Differential release of cellular and scrapie PrP from cellular membranes by Pl specific phospholipase C. Biochem 1990; 29: 5405-5412.

69.Fetcher A. Suspected dimethoate toxicity in cattle. Mod Vet Pract 1984; 65(4): 283-285.

70. De Reuck J, Colardyn J B, Willems J. Fatal encephalopathy in acute poisonings with OP insecticides. Clin Neurol Neurosurg 1979; 81(4): 249-254.

71. Stahl N, Borchelt D R, Hsiao K, Prusiner S B. Scrapie prion protein contains a phosphatidylinositoiglycolipid. Cell 1987; 51:229-240.

72. Domenech C E, de Domenech E E M, Balegno D, de Mendoza D, Farias R N. Pesticides action and membrane fluidity. FEBS Lett 1977; 74: 43.

73. Supernovich C, Crain R, Rosenberg P. Effect of soman and sarin on phosphatidylcholine asymmetry in the electroplax from electric eel. J Neurochem 1991; 57(2): 585-593.

74. Savolainen K M, Hirvonen M R. Second messengers in cholinergic-induced convulsions and neuronal injury. Toxicol Lett 1992; 64/65: 437-445.

75. Shi B, Oswalt M D, Chou K, Hang A. Loss of inositol phospholipids in epididymal sperm following exposure of mice to long term feeding of OP. Pharmacol Toxicol 1992; 90. 70: 75-76.

76. Wuthrich J. Nature 1996; 382: 180.

77. Bolton D C, Bendbeim P E. A modified host protein mode of scrapic. In: Bock G, Marsh J, eds. Ciba Foundation Symposium 135. Chichester: John Wiley, 1988: 164-177.
78. Sagolainen K M, Muona 0, Nelson S R, Samson F E, Pazdernik T L. Lithium modifies convulsions and brain
phosphoinositide turn over induced by 0Ps. Pharmacol Toxicol 1991; 69: 346-354.
79. Patton S E, Lapadula D M, O'Callaghan J P, Miller D B, Abou-Donia M B. Changes in in vitro brain and spinal cord
protein phosphorylation after a single administration of TOCP to hens. J Neurochern 1985; 45: 1567-1577.
80. Bassant M H, Picard M, Olichon D, Cathala F, Court L.Changes in the serotonergic, noradrenergic and dopaminergic
levels in the brain of scrapie-infected rats. Brain Res 1986; 367:360-363.
81. Mathews C. Nucleotide metabolism. In: Van Holde R E, ed. Biochemistry, 2nd edn. Menlo Park, CH: Benjamin
Cummings, 1996: 800-80 1.
82. Baraban J M, Worley P F, Snyder S H. Second messenger systems and psychoactive drug action: Focus on the phos-
phoinositide system and lithium. Am J Psychiatry 1989; 146:1251-1260.
83. Smith S J M, Kocen R S. A CJD-like syndrome due to Lithium toxicity. J Neurol Neurosurg Psychiatry 1988; 51:120-123.
84. Marquis J K. Non cholinergic mechanisms of insecticide toxicity. Trend Pharmacol Sci Feb 1985: 59-60.
85. Koretzky G A. Proc Nad Acad Sci USA 1991; 88: 2037-2041.
86. Waliby M, Shelef L A, Luk G D, Majurndar A P N. The role of tyrosine kinase in the induction of gastric mucosal
cell proliferation by the fungicide captan. Toxicol Lett 1990; 54(213): 189-198.
87. Nogueira A C, Neubert R, Felies A, Jacob-Muller U, Frankus E, Neubert D. Thalidomide derivatives and the immune
system. 6. Effects of two derivatives with no obvious teratogenic potency on the pattern of integrins and other surface receptors on blood cells of marmosets. Life Sci 1995; 58(4): 337-348.
88. Neubert R, Hinz N, Thiel R, Neubert D. Down regulation of adhesion receptors on cells of primate embryos as a probable teratogenic mechanism of thalidomide. Life Sci 1995-96; 58(4): 295-316.
89. Hope J. Update BSE. J Biol Ed 1990; 24: 225-228.

90. Environmental and Molecular mutagenesis. New York: Liss, 1987:20,73,92.

91. Kiraly J, Szentesi 1, Ruzicska M, Czeize A. Chromosome studies in workers producing OP insecticides. Arch Environ Contarn Toxicol 1979; 8: 309-319.

92. Slamenova D, Dusinska M, Gabelova A, Bohusova T, Ruppova K. Decerntione-induced single strand breaks to human DNA, mutations at the hgprt locus of V79 cells, and morphological transformations of embryo cells. Environ Mol Mutagen 1992; 20: 73-78.

93. VIckova V, Miadokova E, Podstavkova S, VIcek D. Mutagenie activity of phosmet. Mutation Research 1993; 302(3): 153-156.

94. Kurinnyi A 1. Comparative study of the cytogenetic effect of certain OP pesticides. Genetika 1975; 11 (12): 64-69.

95. Salmanova D V, Nikitin A M, Zhuze A L, Gurskii G V, Zesedatelev A S. Interaction of synthetic ligands containing NA-disubstituted mono- and diphthalimide with DNA. Mol Biol Mosk 1995; 29(4): 848-861.

96. Hall 1 H, Wong 0 T, Scovill JP. The cytotoxicity of Npyridinyl and N-quinolinyl substituted derivates of phthalimide and succinimide. Biomed Pharmacother 1995; 5: 251-258.

97. Crombie T, Swaffield J C, Brown A JP. Protein folding within the cell is influenced by controlled rates of polypeptide elongation. J Mol Biol 1992; 228: 7-12.

98. Dallocchio F, Matteuzzi, Bellini T. Non enzymic protein phosphorylation. Biochem J 1982; 203: 401~404.
99. Wali R K. Subchronic malathion treatment effects upon rat intestinal function. Bull Environ Toxicol 1984; 33: 289-294.
100.Ecobichon D J. Pesticides and neurological disease. Boca Raton, FL: CRC Press 1982: 181.
101.Dambska M. Blood brain barrier in young rabbit brain after dichlorvos intoxication. Neuropatol Pol 1984; 22: 129-137.

102. Clouscard C, Schelcher F, Beaudry P et al. Different allelic effects of the codons 136-171 of the prion protein gene in sheep with natural scrapie. J Gen Virol 1995; 76(8): 2097-2101.

103. Kurian SS, Ferri G L, De Mey J, Polak J M. Immunocytochemistry of serotonin containing nerves in the human gut. Histochernistry 1983; 78: 523-529.

104. Sly J M A. (Agricultural Development and Advisory Service, Hatching Green, Harpenden, Herts.) Review of usage of pesticides in agriculture and horticulture in the UK; Report No 41 and 23; Feb 1985 and Jan 1982. London: MAFF Publications.

Back to Purdey Home Page