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High Dose

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Scientific papers - High dose - page 7

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.

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