An Introductory Hypothesis on the causes of the Transmissible Spongiform Encephalopathies
The Transmissible Spongiform Encephalopathies (TSEs) comprise a variety of neurodegenerative diseases such as Bovine Spongiform Encephalopathy (BSE) in cattle, Creutzfeldt-Jacob disease(CJD) and variant CJD (vCJD), Gerstmann Straussler Syndrome(GSS) and Kuru in humans, Scrapie in sheep, Chronic Wasting Disease(CWD) in deer and elk, Feline Spongiform Encephalopathy (FSE) in cats. We believe that sporadic TSEs are multifactorial diseases, with a genetic component, caused primarily by a specific configuration of Environmental factors that give rise to abnormalities in the cellular manufacture of the hosts Prion protein. The host has problems in handling the type of electron movements generated by oxidising agents, due to an altered configuration of trace metals such as Copper(Cu), Manganese (Mn) and their interaction with a 'partner' protein, known as the prion. The most common pre requisites are - 1) High Manganese (Mn) levels 2) Low copper(Cu) and anti oxidant levels 3) High levels of oxidising agents. There is no requirement for the introduction of an 'infectious' agent to the host.
It is now widely accepted that a metallo-protein, richly expressed in the nervous system, called the Prion, in it's abnormal form is central to the disease pathology. This protein is normally assembled within the cell from amino acids and 2 or 3 Cu atoms that bind in to histidine residues of the octapeptide repeat region of the protein. The prion moves to the outer surface of the cell membrane where it becomes attached by a glycolipid anchor for a few hours, before being drawn back into the cells lysosomes. Its then broken down by protease enzymes into its constituent amino acids for recycling.
Normal prion protein can function as an anti oxidant - Super Oxide Dismutase (SOD)(1) and its well positioned on the cell surface to protect nerve cell membranes from free radical damage Cu acting as a 'sink' for stray electrons. It also plays a role in the transport of Cu in and out of cells, providing Cu for nerve transmission at the synapse and redistributing Cu to protect against toxicity/deficiency.There are also, probably, other roles yet to be described. Its prevalence in the retina and skin suggest there may be a function in dealing with electron transfer and acceptance connected with light energy.
When the Mn levels are high and Cu low, the cell cannot manufacture the normal Cu form of the protein. The protein binds Mn as a second choice substitute. At some point in the protein's life cycle, probably when it is in position on the cell membrane, the protein misfolds into an abnormal shape(conformational change). We think the cause of this is increased oxidative stress which changes the charge on the Mn atoms.The prions are also now unable to perform their crucial role as anti oxidants and in the transport of Cu, which exacerbates the situation. In this abnormal form the prions are insoluble in the protease enzymes of the lysosomes and consequently they build up in the cell, which eventually bursts. The abnormal proteins then aggregate, possibly due to abnormal electrical charge, and form the characteristic metal rich fibrils of TSEs. This has now been demonstrated in living cell cultures by Dr David Brown et al at Cambridge University(4)
FUNCTION OF PRION PROTEIN
The normal Prion
Proposed function of normal cellular prion protein; N terminal cleavage of the membrane anchored prion protein releases the octapeptide Cu domain segment into extracellular space to patrol as a scavenger / quencher of excess endogenous porphyrin/xeno photosensitiser molecules.
The abnormal prion
Proposed dysfunction of pathogenic prooxidant Mn3+ prion, Mn loaded / Cu depleted PrP fails to scavenge/quench the charge emitted by hyper photoexcited xenophotosensitiser and Mn prions, thereby subjecting the CNS to a lethal prooxidant melt down.
A non transmissible type of Spongiform Encephalopathy (SE) can be produced by exposing animals to a chemical that lowers(chelates) copper. This follows the experimental model set up in the late 60s by I.H.Pattison et al.(3) and is an early precedent for the idea that a specific abnormal cellular chemistry can create a terminal prion disease in animals.
Studies of environments in which TSEs flourish show high levels of Mn and low levels of Cu and antioxidants (2) and this would, to some extent, be reflected in the inner cytosolic environment. High levels of Mn (10x) have also recently been found in CJD brain tissue examined by Dr Brown . Homeostatic mechanisms, determined by genotype, would try to even out metal imbalance, but are only partially effective in extreme locations and vary from animal to animal.The higher the Mn levels the greater the take up by prions.
This abnormal prion material has a transmissible pathogenic potential in certain circumstances, which we regard as secondary, and very rare in life.Transmission studies show that intracerebral or intravenous innoculation or feeding with brain homogenate can infect experimental animals. However this is not sufficient to demonstrate Kochs postulates because it doesnt replicate exactly what occurs in life.(Animals /humans don't eat homogenate) The homogenisation process is very aggressive and chemically alters the brain material by freeing up protein bound reactive metals from their proteins. In this form there is a greatly increased capacity to generate toxic free radicals(12). The material is then used in concentrated form to infect animals.This cannot be considered a reliable predictor of in vivo oral infectivity particularly in a disease in which free radicals and divalent cations (Mn,Iron,Cu) are a central feature.
The characteristic of transmissibility would however be important when considering blood transfusions , as many blood cells express the prion protein and there is no additional protection from a 'species barrier'. If white blood cell prion had changed into it's abnormal form it could pass on CJD ( Leucodepletion - removal of white cells from blood stock is practised in the UK for this reason) It seems feasible that the capacity forinfectivity is contained in the clumps of protein plaques that are rich in reactive metals such as Mn and Iron.. This neurotoxic potential lies in a capacity to kick start a lethal free radical, prion specific pathway, as the prion fails to carry out normal electron shuttling on the cell membrane. Finally the mutant prion acts as a powerful pro -oxidant in its own right and leads, via build up of surplus electrons, to the manufacture of excessive quantities of toxic by-products such as hydroxyl radicals, hydrogen peroxide, peroxynitrite or quinones that would eventually destroy the neurones. This closely resembles the mechanisms of TSE's 'sibling' disease, Alzheimer's disease.
The official theories are very vague and I am not aware of one clearly stated and referenced exposition. The UK Ministry of Agriculture (MAFF) cites abnormal prion material of ovine origin, contained within Meat and bonemeal feed (Mbm) as the cause of BSE. There is a lot of scientific contra evidence to this idea. There has been £140 million spent over 16 years and yet it still can't demonstrate Koch's postulates.
BSE is caused by a number of factors.
1) Organophosphates (OPs). The epidemiology of BSE shows a strong correlation between high dose, systemic, phosmet use and BSE (10) A number of possible mechanisms for this have been proposed.(10, 11, 12). Five years prior to BSE a high dose (4x max recommended dose) lipophilic, systemic formulation of the di thiophosphate OP pesticide, Phosmet, was made compulsory, in the UK only, to treat cattle for warble fly. This was poured along the cows spine, millimeters from the target organ, the spinal cord. This was usually a bi annual treatment with a voluntary follow up dose at 2x recommended max. dose. Also, due to the changes in the rendering process at this time the residues of Phosmet and other pesticides in Mbm increased (6,12). Phosmet was also being used at this time on a voluntary basis at 2x max dose as a powder to control lice.
Firstly the 2 sulphurs in a di- thiophosphate type of OP,such as phosmet, bind to Cu atoms forming a mercaptide ring, effectively lowering Cu in the cells. (Viewed in the perspective of the SE induced by the Cu chelator cuprizone, mentioned above, BSE would not seem to be such a surprising outcome).
Secondly, as Phosmet exerts OS, it up regulates (up to 10 fold) prion protein, even at doses as low as 2 parts per million.(7) This latter attribute, at the very least, would have amplified the host production of abnormal prions whatever the trigger. This would have reduced the incubation times sufficiently to precipitate an epidemic. The up-regulation would also greatly increase the available prion Cu binding sites in the cell, at the same time as reducing the available Cu. The protein would need to bind a second choice metal at these sites to enable it to fold.
Thirdly phosmet's oxidative potential could be oxidising the prion bound Mn on the cell surface to Mn3+ or 4+ locking in the pathogenic conformation which in turn blocks the sites available to degradation enzymes - the host's only defense.
Other OPs that don't 'lock up' Cu pose less risk for TSE, but they do all act as oxidising agents and risk would increase in systemic formulations applied close to the eyes, brain and spinal cord and as the dose is increased.
In France a similar pattern is now starting to emerge since they have embarked on their own 'varron' ( similar to the Warble fly) eradication campaign. They are using Phosmet in an oil based formulation and Ivomectin. Professor Michel Bounias has been researching this and has found a strong correlation between the spread of BSE and this campaign. The UK exported feed to France from1988 to 90 and if this was to blame you would expect to see cases starting in France around 92 and falling from the mid nineties.You don't. The pattern is of a continuous rise with estimates putting the number of BSE cases at between 1200 and 7000.
Can Phosmet by itself cause Spongiform Encephalopathy? This is an intriguing question and it cannot be answered yet for sure. My view is that it can, if the Mn level is relatively high in cows (perhaps still within the reference range) and further research is needed to prove this. Whatley et al showed that it produced 4 out of 5 of the key changes required for TSE.(7) Protease resistance was not clearly demonstrated in these tests but this may have been because there was no Mn in this type of cell culture.The copper levels were normal.
2) Manganese and copper. There were a number of sources of raised Mn that would have affected cattle in the early 80s. Mbm contained high residues after the addition of chicken manure -chickens had been fed high doses of Mn to strengthen egg shells most of which was excreted; Mn supplemental licks, and powders were sometimes added to feed troughs. The increasing use of Mn containing pesticides such as Maneb and Mancozeb(2); Mn levels in grass are naturally high in many of the BSE zones. It seems likely that the abundant Mn bound to the vacant Cu sites of the prion as the cells failed to source enough Cu to meet the demands of raised OS.
Another possibility is a Mn compound such as Mn
oxide or Mn silicate is involved. Mn-oxides often occur in the
Environment in highly concentrated nodules. One detailed study on
a N.Devon farm (8) found the Mn minerals varied considerably over
the farm but in one area the nodules contained high
Mn and virtually undetectable Cu (and Cobalt). Also common in the
nodules were reactive metals such as Aluminium and Iron. This
area is part of a Scrapie belt. in N.Devon and Somerset. Mn is
also thought to exist rarely in nature in its more highly
oxidised, toxic form, Mn3+ in pyroxene minerals.
Another interesting study found high levels of Mn oxides deposited in the upper sand layer of a water treatment plant in Grampian. These oxides adsorbed many of the other metals that were being filtered out in the treatment process (9). On a research trip to Colorado Mark found a cluster of CWD cases confined to deer that were drinking from the immediate outfall of a water treatment plant. These are both cases that we will be researching further
3) Oxidative Stress(OS). If the cell is failing to deal with OS due to exposure to a free radical trigger and misfolding of prion protein then the Mn species that the prion binds may be one of the more reactive and toxic, highly oxidised types, such as Mn 3+, 4+or 7+. Alternatively oxidation occurs on the cell membrane the prion having bound Mn 2+ , the subsequent loss of electrons causing a refolding of the end region of the protein. Common sources of OS that may be relevant are pesticides and ultra violet light.
4) When abnormal cattle prion reached Mbm this may also have amplified the disease, if it was able to pass through a leaky gut wall into the blood stream or into the lymph via a gut interface such as Peyers patches.
We think this is caused by exposure to similar factors as produced BSE. ie a source of reactive Mn, low Cu and raised OS possibly from infrasound, pesticides or ultra violet light. The argument that because BSE and vCJD are pathologically almost identical doesn't prove a cause and effect between BSE to vCJD. If you discovered a pathology of lead poisoning in a cow and its herdsman would this prove that the cow had transmitted the pathology to the herdsman or vica versa or could they both have been exposed to a common trigger.
The epidemiology of vCJD and sporadic CJD very strongly suggests that environmental factors common to victims are at work, because many of the cases are very close together, 200 metres in some cases. Abnormal bovine prion must have been so common in the late 80s in a wide variety of processed foods consumed all over the UK, that virtually the whole population has been exposed. Its thus very difficult to understand why this alleged ubiquitous 'infectivity' should concentrate CJD in precise topographical foci. In Queniborough, nr Leicester, for example, there has been one case of classical CJD (12 years ago ) and 2 recent cases of vCJD -all 3 in the same road, and 3 further vCJD cases in the area and a case of Feline Spongiform Encephalopathy.
What we found in the area was high to very high soil Mn levels; spraying of sewage sludge, which contains high levels of reactive metals (ie Mn, zinc, cadmium) ; an interesting industrial and building history - A munnitions dump that had been built over in the fifties/ earth moving ; pollution events from a, now closed, dye works ( some dyes use Mn) on the local industrial estate (there are many reports of dusts/sands over a long period of time.). There were also 11 annecdotely reported cases of ME/CFS in one road in the village.
Another cluster is at Armthorpe, East Doncaster, an old pit village which has very high Mn soil levels.There have been 3 cases of vCJD in one road here.Close by I found a water treatment plant and extensive excavations for roads and new industry.Walking around the area there was a lot of airborne dust/sand which got in my eye and throat. I sampled both excavation soil and natural ground - the Mn level in excavations was double that of natural ground in my survey.
Adswood, South Manchester has had 2 cases 200Metres apart.
A number of the early cases of vCJD and BSE occured in a ribbon of very low population density in mid Kent. Early BSE occurred at Plurenden Manor Farm in 1985, with 1 case of vCJD in High Halden and 1 in Bethersden, both a mile away from the farm.Two local pollution incidents are of interest here - the Smarden spill (1959 on) which involved OPs from a manufacturing plant contaminating local land and a leak at an ICI chemical plant at Yalding in April 1986 - described as a toxic fog. Pesticides are also used intensively in this area which is known as the 'Garden of England.'
There are several known mutations on the gene that encodes for prion protein, and these influence the progression and pathology of the disease. GSS is an inherited human prion disease which is fatal to carriers of the mutant gene. Most of the other associated mutations do not necessarily result in death from a prion disease.
Last year we studied the very high incidence of CJD that is occurring in a small village in Southern Italy, amongst the Greek population. This began in the mid nineties. All the 20 victims were carriers of a prion gene mutation (E 200K) There have been no cases in all the other Greek carriers scattered throughout a wide diaspora in Italy. Preliminary studies revealed high soil Mn, low Cu, the use of di thiophosphates on Bergamots, some local industries that would present risks for CJD - Dye works. This strongly suggests an environmental trigger and resembles the situation that we studied in Slovakia last year.
We think that genetics is relevant beyond the prion gene because poor/ altered gene expression for a number of enzymes and metallo proteins would be an added risk factor in these diseases if we are correct.
This hypothesis is based on several worldwide Environmental studies with lab analysis; direct research in cell culture; epidemiological analysis and extensive review of a very wide Scientific literature. Much of it is beyond reasonable doubt but some of it should be repeated and extended at other labs for confirmation. Obviously much new work needs to be done.We believe we should identify and follow up all reasonable risk factors. Many of them, in the fullness of time, will no doubt turn out to be red herrings but at this stage you have to work through everything to find the truth. You also have to be prepared to adapt and jettison your ideas if they don't stand up to scientific scrutiny.
We're a long way from having all the answers to TSEs but this is not surprising as they are complex, multifactorial diseases like Alzheimers. To cast them in the mould of simple infectious diseases and to generate preventive measures from this premise, however convenient, is simply wrong: This is why BSE is rising in so many countries. Unfortunately the vast body of experimental work using homogenate, which did demonstrate a theoretical infectious risk, deceived scientists into focusing on a 'prime suspect' too early. The way things are going at the moment, it looks as if they'll keep chasing this until the cows come home.
1) Brown D.R. and Besinger A. Prion protein expression and superoxide dismutase activity. Biochem J. 1998; 334 423-429
2) Purdey M. Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical generating divalent cation Manganese. Medical Hypotheses(2000) 54(2) 278-306 click to download complete text
3) Pattison.I.H, Jebbett J.N. Spongiform encephalopathy induced in rats and guinea pigs by cuprizone. Exp. Mol.Pathol. 1969; 10 (3) 274- 287
4) Brown D R.et al. Consequences of manganese replacement of copper for prion protein function and proteinase resistance..The EMBO Journal Vol 19 No 6 pp 1180-1186. 2000. Full text : http://www.emboj.org/cgi/content/full/19/6/1180?ijkey=sBpvL9/Ho3r0A
5) Halliwell B.and Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford: Clarendon Press 1989.
6) Wilesmith J.W. et al; Bovine Spongiform Encephalopathy: Epidemiological studies on the origin. The Vetinary Record , March 2, 1991; 128, 199-203
7) Whatley S.A.et al Phosmet induces up-regulation of the surface expression of prion protein. Neuroreport May 1998
8) Lideard, H.M. et al 1993 Identification of Mn-oxide minerals in some soils from Devon ,UK,and their varying capacity to adsorb Co and Cu. Environmental Geochemistry and Health 1993 15 (2/3) 93-104
9) Eley, M and Nicholson, K. 1993 Chemistry and adsorption-desorption properties of manganese oxides deposited in Forehill Water Treatment Plant, Grampian, Scotland. Environmental Geochemistry and Health 15 (2/3) 85-91
10) Purdey, M. Are Organo-phosphate pesticides involved in the causation of BSE? Hypothesis based upon a literature review and limited trials on BSE cattle. J.of Nutritional Medicine 1994; 4(1) 43-82.
11) Purdey, M. The UK epidemic of BSE ; Slow virus or chronic pesticide initiated modification of the prion protein? (parts 1 and 2) Medical Hypotheses 1996 . 46: 429-454
12) Purdey, M. High dose exposure to systemic phosmet insecticide modifies the phophatidylinositol anchor on the prion protein: the origins of new variant transmissible spongiform encephalopathies? Medical Hypotheses 1998 50, 91 - 111
General background on Manganese - Aschner M, Aschner JL: Manganese neurotoxicity ; cellular effects and blood brain barrier transport. Neurosci Biobehavioral Revs 1991; 15 333-340
Oxidative stress and free radicals see (5)
© Nigel Purdey : April 2000