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Medical Hypotheses (2001) 57(1), 29-45
2001 Harcourt Publishers Ltd doi: 10.1 054/mehy.2001.1305, also available online at http://www.idealibrary.com on

Does an ultra violet photooxidation of the manganese loaded copper depleted prion protein in the retina initiate the pathogenesis of TSE?

M. Purdey
Elworthy, Taunton, Somerset, UK.

Summary. Ecosystems supporting clusters of sporadic transmissible spongiform encephalopathy (TSE) are characterized by common properties of high-manganese/low-copper, zinc, selenium mineral status, and highaltitude/snow-covered/pre-cambrian mountain terrain where above-average intensities of ultra violet/ozone oxidants are prevalent. Cell culture trials have confirmed the hypothesis that manganese (Mn) substitutes at Prion Protein's (PrP's) vacated copper (Cu) domain, whereupon PrP loses its Cu-mediated antioxidant function, transforming into a protease-resistant misfolded isoform that aggregates into fibril 'tombstone' structures - the key hallmark distinguishing TSE central nervous system (CNS) pathology. The cellular localisation of PrP suggests PrP serves a 'front line' contributory role in neutralizing radicals generated by incoming environmental oxidants, whilst an intensive expression of PrP messenger ribonucleic acid (mRNA) in the retina, melanocytes, epidermis, etc., suggests PrP performs a key antioxidant role as a 'photooxidative shock absorber'; binding of porphyrin IX, Congo red and other photosensitisers to PrPc suggests PrPc serves as an integral associate of the porphyrin/melanin chromophore electron transfer chain; thereby serving as a quencher of singlet O~superoxide generated by photoenergised chromophores/xeno photosensitisers. It is proposed that sporadic TSE pathogenesis is initiated in the retina of environmentally/genetically predisposed individuals via a two-stage chronic toxic process - Mn substitution at PrP's Cu domain forming a stable Mn2+-PrP complex, followed by an ultra violet in situ photo-oxidization of the Mn2+ component; whereby the latent 'Jekyll and Hyde'capacity of the Mn2+-PrP conjugate is activated into the fully fledged, 'infectious' lethal autooxidizing, Mn3+-PrP 'prion' agent. Thus, PrPc's Cu-mediated antioxidant function is replaced by a Mn3+-mediated autooxidant dysfunction. Could the UK's increased loading of a cocktail of environmental oxidants that penetrated the CNS of the UK bovine (ultra violet microwaves/ozone/systemic cu-chelating insecticides) account for a more virulent Mn4+ mediated acceleration of the TSE degenerative process in Mn-contaminated/genetically predisposed individuals, manifesting as the widespread emergence of new-variant bovine spongiform encephalopathy (BSE), variant Creutzfeldt-Jacob disease (vCJD)/FSE in younger mammals? Copyright. 2001 Harcourt Publishers Ltd.

Received 20 December 2000, Accepted 9 January 2001
Correspondence to: Mark Purdey, E mail: MadCowPurdey@aol.com

INTRODUCTION
The conventional hypothesis on the origins of new variant (nv)/sporadic TSEs considers that ingestion of TSE-infected CNS material (meat and bone meal rations, bovinederived baby foods, scrapie-diseased sheep's brain, etc.) is sufficient to initiate TSE in PrP-susceptible genotypes (1p,2). However, the proponents of this theory do not address the fact that significant tonnages of the 'nvTSE contaminated' foods which were incriminated in the UK's BSE/nvCJD epidemics were exported worldwide to bovine/human populations that remain free of nvTSE epidemics (3). Nor do they address the fact that BSE has failed to emerge in various TSE-susceptible species (e.g. sheep, goats) (1,2) who were fed the same TSE-incriminated feed.
These flaws in the conventional theory (4) suggest that TSE-contaminated feed could either play no role in TSE aetiology or it could play a contributory role in conjunction with the genetic/environmental causal prerequisites that are prevalent in the environments where TSE epidemics erupt.
An alternative theory has evolved which expounds the possibility that TSE pathogenesis is initiated in susceptible individuals who are chronically exposed to a double- barrel' combination of specific environmental properties that have been identified as common to TSE cluster foci worldwide (4).
Whilst endorsing the mainstay of S. Prusiner's 'prion' hypothesis (2), which heralds the conversion of a native nerve membrane glycoprotein, prion protein (PrPc), into a misfolded, protease-resistant, 'infectious' isoform (PrPsc) as integral to the development of TSE, this theory extends Prusiner's concept by citing novel laboratory (5)/field trial (4) data that amass a strong case for an 'environmental origin' hypothesis as an explanation of the primary initiation of CNS PrP conversion and the ensuing development of TSE.

A two stage toxic pathogenic mechanism initiates TSE
A toxic template for TSE aetiology is proposed which is based upon chronic exposure of susceptible individuals to environments which are characterised by a high manganese (Mn)/low-copper (Cu), selenium (Se), zinc (Zn) mineral status and above-average levels of photooxidative /ozone-oxidative stress.
The putative pathogenic mechanism hinges upon a switch in PrPc's molecular conformation, wherein PrPc loses its normal Cu-mediated antioxidant function (6) and adopts a lethal prooxidant dysfunction. This twostage pathogenic process primarily entails an abnormal Mn substitution at the vacated Cu domain on PrP, causing PrP to misfold into a stable protease resistant Mn2+PrP isoform (5), thereby losing its specific Cu-mediated antioxidant function.
The second pathogenic step involves an in situ. ultra violet (or other central nerve-penetrating environmental oxidant; ozone, systemic insecticide, radio transponder collar/mobile phone, etc.) mediated oxidization of the Mn component of the Mn2+-PrP conjugate in the mitochondria; where the innocuous 'dormant' Mn2+-PrP complex is activated into a lethal 'infectious" auto-oxidizing Mn3+/Mn4+-PrP stable pathogenic agent (see Fig. 1).
The low levels of Se/Zn/Cu that have been found to characterize the TSE cluster environments (4) would further predispose mammals that were self-sufficient upon these foodchains to low-antioxidant activities of the Cu/Zn-dependent superoxide dismutases (SODs)/catalases and Se-dependent glutathione peroxidases (7,8), thus permitting the radical chain reactions resulting from Mn3 +/Mn4+ -initiated autooxidation (7,9,10) to proliferate out of control. Such auto-oxidizing initiated cascades rapidly spread and envelop many of the mitochondria-rich tissues/cell types of the CNS where Mn is highly concentrated (the pituitary, pineal, basal ganglia, astrocyte cells, etc.) (11,12), thereby subjecting surrounding membranes and tissues to the chaotic pathogenic assault of lipid peroxidation and oxidative self-destruction (7,8).

The two stage toxic pathway of sporadic TSE pathogenesis in the retina.

The two stage toxic pathway of new variant BSE pathogenesis in the central nerves.
Fig. 1 The two stage toxic pathway of new variant BSE pathogenesis in the central nerves.

The 'Jekyll and Hyde' redox status of the Mn-prion conjugate
Mn 2+ normally exerts an important antioxidant function whilst harnessed to the active site of the Mn SOD enzyme expressed in the lung, liver and CNS (7). Mn can also exert abnormal prooxidant activity which is usually associated with toxic CNS accumulations of Mn once the metal has been oxidized into its trivalent Mn3+ oxidative species (9) - a situation that is more prevalent in genotypes who are unable to express adequate levels of Mn SOD activity.
In respect of the 'Jekyll and Hyde'-like redox status that pertains to Mn within biological systems, the incorporation of Mn in the structure of the melanin chromophore (10,13) may explain the mystery surrounding this same alternating redox property of melanin and how this chromophore can metamorphose into a potent generator of photoinduced oxygen species that induces melanoma (14), having lost its normal protective function as a quencher of photoinduced oxygen radical.
This theory ascribes a similar 'Jekyll and Hyde' property to PrP, where TSE pathogenesis is initiated once the vacant Cu domain on PrP (15, 16) has acquired an abnormal Mn3+/Mn4+ -mediated autooxidant malfunction (7,9) having lost its normal Cu-mediated antioxidant function (6).

The relationship between Mn species and TSE 'strain' types
Mn substitution at PrPs Cu domain could represent the primary pathogenic cornerstone that is fundamental to the aetiology of most 'strain' types of TSE. Substitution could also occur with other hitherto unidentified transition metals that are able to ligate at PrPs octapeptide Cu domain.
However, other TSE straintypes could develop following a range of pathogenic scenarios that all result in a common disturbance at PrP's Cu domain leading to a loss of PrP's Cu-mediated antioxidant function. One such mechanism could involve a pollutant induced lipid peroxidation of membranes, where the resulting peroxides interact at PrPs Cu2+ domain initiating an 'in situ' fomation of hydroxyl/CU3+ radicals (7).
The specific strain type of TSE emerging at the end of the day is dictated by the specific valency of the oxidized Mn species conjugated to PrP, coupled to the PrP genotype (1,2) of the exposed individual. For instance, Mn3+PrP could represent the causal agent responsible for sporadic TSEs, whereas the more potent pro-oxidant Mn4+-PrP could underlie the aetiology of nvTSEs.
These factors will influence key criteria that allow distinction between the different strains of TSE, such as speed of incubation period, the distribution of lesions in the CNS, etc. (17).

Mn bioaccumulation and the Mn-prion pyramid
The primary initiation event of this environmental hypothesis can operate either singly or in conjunction with the consumption of TSE-contaminated feeds; where first-stage initiation of the 'dormant' protease-resistant Mrln2+-PrP complex can either be generated via an endogenous de novo self-assembly in the CNS of susceptible genotypes who have been chronically exposed to high-Mn/Low-Cu environments, or acquired via ingestion (or vaccination) of an exogenous contaminant of feed derived from Mn2+-PrP 'infected' CNS material; that Mn2+-PrP having been generated and carried as 'dormant' within previous generations.
The model of a 'Mn-prion pyramid' (Fig. 2) is employed to depict the increasing CNS concentrations of protease resistant Mn-prions which are bioconcentrated up the farm animal food chain whenever practices of cannibalistic consumption of prion-contaminated CNS material from preceeding generations are implemented, e.g. in kuru (18) and BSE (2) type TSEs.

Fig. 2. Manganese 3+ prion pyramid of bioconcentration in the development of kuru tse in fore tribesfolk of Papua, New Guinea following intake of volcanic Mn Oxide contaminants via local food/air combined with cannibalistic ingestion of Mn rich CNS/pituitary tissues.

Recent trials by Collinge et al. (19) have demonstrated that prions can exist in two forms in vivo: as an innocuous dormant form and as an active TSE-generating form. This theory proposes that the introduction of an exogenous oxidizing agent which oxidizes the Mn2+ component of the dormant PrP complex into the lethal auto-oxidizing MW+-PrP complex, provides a feasible candidate mechanism that can explain the insidious transformation process operating between the two forms as well as its relationship to the ultimate emergence of clinical TSE

EVIDENCE FOR A HIGH-Min/LOW-Cu, Se, Zn IN FOOD CHAINS OF TSE FOCI
Analyses of foodchains supporting high-incidence focal 'clusters' of sporadic TSE (CJD in Slovakia, Calabria scrapie in N. Iceland and Chronic Wasting disease (CWD in Colorado, USA) nv CJD in Queniborough, Armthorpe has consistently demonstrated more than a 2.5 times higher concentration of Mn in relation to levels recorded in adjoining TSE-free localities (4). Likewise, levels of Cu, Zn and Se that serve as antioxidant cofactors in biological systems were consistently classed as very low in all the sporadic TSE regions studied (4).
The source of Mn contamination in these cluster zones originates from a variety of natural (volcano) and industrial (steel, glass, munitions, lead-free fuel, dye, etc.) manufacturing contexts where Mn had been combusted into an oxide/silicate form and vented into the surrounding environment as an airborn pollutant (4).
Interestingly, environments where certain types of Mn oxide mineral (e.g. Lithiophorite) are naturally prevalent in the soil are also very deficient in Cu/Zn/Co, because of the capacity of these Mn oxide minerals to adsorb and conjugate with these trace elements (20). Such a geochemical mechanism may elucidate the underlying basis for the existence of this specific abnormal mineral configuration that has been identified in TSE foodchains (4), and furthermore elucidate the fundamental in vivo mechanism through which manganese oxide induces TSE pathogenesis in the biological system.
Long-standing high-incidence clusters of TSE were selected for analytical research where the respective TSE affected populations are self-sufficient upon their local food chain (4). Evidence for the presence of an environmental causal factor in TSE cluster zones is demonstrated by the repeated failure of blanket slaughter regimes executed for controlling animal TSEs across many of these zones (22,1,2).

COLORADO CLUSTER OF CHRONIC WASTING DISEASE IN CERVIDAE
Along a 100-mile stretch of the Front Range of the Rocky Mountains in N. Colorado, 7% of the deer and 2% of the elk are affected with the TSE chronic wasting disease (CWD) (21,22). Cervidae are overstocked in this region around the Rocky Mountain National Park and Fort Collins, and, according to local ranchers and wildlife wardens, the deer have acquired a taste for consuming pine needles as a dietary replacement for their meagre food supply. Toxic levels of Mn have been recorded at levels up to 1886 p.p.m. in the pine needles of this region (23) and more generally in soil matrices (4), which could be related to the increased acidification of rainfall falling along the Front Range rainbelt zone (24) as a result of airborne pollutants arising from the nearbye Denver industrial conurbations to the south-east (25). These pollutants are widely recognized to blow onto the Front Range ridges via the prevailing air currents, whereby the warm air is forced to rise up the foothills and rapidly cool - the pollutant particulates falling back to earth in the rainfall. Increased soil acidity unlocks certain soil cations, such as Mn and aluminium (AI), rendering them more available for uptake into the vegetation horizon above (26)

.
SPORADIC / FAMILIAL CJD CLUSTERS IN SLOVAKIA
Sporadic/Familial CJD runs at a high 1 per 1000 head incidence rate amongst the residents of a group of neigh- bouring villages (Zuberec, Malatina, etc.) on the western slopes of the High Tatra mountains in Slovakia (27-29). Scrapie is also known to affect the local sheep flocks (30). Analysis has identified toxic levels of Mn in various plant matrices and human hair throughout the CJD endemic environment in relation to adjoining CJD-free controls (4). The CJD foci lie approx 10 km downwind of several ferromanganese and a television component plants sited along the Orava valley (4). Emissions were largely unregulated during the Communist era, which resulted in serious 'die back' of the local pine trees - particularly prevalent around the villages affected by CJD (unpublished survey by V1adimira Popikova). Mn was recorded at 951 p.p.m. in pine needles of the CJD endemic environs and only 59 p.p.m. in adjoining CjD-free locality (4). Popikova's survey in the economically deprived vicinity of Zuberec revealed a unique dietary custom involving the consumption of pine needles as the key ingredient of a home-made 'syrup' and 'tea' *
Interestingly, two CJD cases contained within a cluster of six cases in Burlington, Ontario, Canada (3 1) were immigrants who had originated from the Orava CJD cluster region of Slovakia. Burlington is commonly referred to as 'Steel City' because of the prevalence of steel factories in that vicinity.

SCRAPIE CLUSTERS IN SHEEP FLOCKS OF N. ICELAND
The highest international incidence rate of scrapie can be found in sheep flocks (up to 90% scrapie-affected) which chiefly reside in certain wetter valleys of N. Iceland around Akureyri (32,33). A consistent 2.5 times higher concentration of Mn (200 mg/kg on average) was recorded in the pasture herbage of the scrapie endem ic valleys in relation to the scrapie-free valleys (80 mg/kg on average) (4).
Mn was presumed to originate from emissions of nearby volcanic eruptions which contaminated herbage 'down wind', whilst the acidic fallout from the eruptions would assist in unlocking the availability of soil Mn. However, the greatly reduced levels of available Mn recorded in the soil in relation to the herbage indicated that Mn contamination originated from an airborne/precipitation route (4).

CJD CLUSTER IN A RURAL HAMLET IN CALABRIA, S. ITALY
Since 1995, a cluster of 20 cases of CJD has erupted amongst a community of rare TSE-susceptible Greek-Italian genotypes who reside in a hamlet (142 000 M2) located in the south of Calabria in Italy (34). The majority of these cases involved part-time small farmers who also worked locally at a railway train/ carriage repair factory where arc-welding of manganese enriched steel is carried out. This type of occupational exposure provides a well-known avenue of exposure to substantial doses of airborne Mn (35), where the metal can be absorbed directly into the brain via the inhalatory-nasal-olfactory route (36)

Fig. 3 The increased manganese contamination and hyperoxidative assault of the UK bovine; the aetiological template underpinning the pathogenesis of new strain TSE-BSE.?

Other CJD victims had worked at a clothing factory where various chemical dyes containing manganese and other metals are handled. Cultivation and ingestion of significant quantities of Mn-rich bergamot oranges and cacti fruit is practised exclusively in this precise region of Italy. The very poor socioeconomic status of this Greek-Italian community has lead them to opportunistically ingest unusually large amounts of these local foods. Soils sampled from the CJD victims' gardens demonstrated high Mn (71 mg/L) and very low Cu (1.7 mg/L) (Table 1).

BSE/ nvCJD EPIDEMIC IN THE UK
'Ihe UK bovine was exposed to increased levels of Mn a from the late 1970s to the mid-1990s (see Fig. 3). Mn oxide was added to concentrated feeds destined for cats, zoo animals, calves and cattle at inclusion rates ranging between 80 and 120 mg/kg (37). Calves also received artificial milk powder containing 3500 ppm Mn - a risky practise applied at an early developmental stage when the animal's blood brain barrier is immature and largely unable to regulate the homeostatis Mn uptake into the CNS (9).
Levels as high as 5000 mg/kg of Mn oxide were added to free-access cattle mineral licks in all Europeon countries with endemic BSE. This could pose a significant hazard to any PrP/MnSOD-susceptible cattle in the herd which develop an idiosyncratic 'fetish' for licking at the mineral trough. Such a level of Mn inclusion considerably exceeded the maximum permitted inclusion rate for Mn of 250 mg/kg laid down in the feedstuff regulations (37).
After 1975, it became fashionable for mineral feed companies to chelate Mn with amino acids for ultraefficient absorption into the animal. Mn oxide was also sprayed in chelated form as a fertilizer onto many fodder crops, etc. growing in Mn-deficient alkaline sandy and peaty soils, etc.
Poultry manure was also added to cattle concentrate feeds up until 1992 in the UK (38). Poultry are fed high 250 mg/kg doses of Mn, because, unlike ruminants, their single stomachs can only absorb 2% of the metal (37). Ninety-eight percent of the Mn contained in poultry feed is therefore excreted in the manure which was routinely dried, pelleted and fed to UK cows. Another practice that could have posed a problem of chronic Mn contamination in the UK bovine was the occasional inclusion of extracted fat sourced from the human sewage system in cattle feeds (sewage is high (44) in Mn).
Whilst increases in acid rainfall (26) have served to unleash an increased supply of available Mn into the human/bovine water supplies (39) and foodchain over recent years, increases in atmospheric Mn have also become a significant reality in the UK. Mn particulates are suspended in the atmospheric drift stemming from the widespread, high-ranking use of Mn-based fungicides (maneb and mancozeb) (40,41,42,4) and sewage/Mn fertilizer crop sprays applied over the majority of the UKs agricultural areas during the 1980s and 1990s.
Interestingly, mini-clusters of vCJD have emerged in specific rural regions or villages/small towns in the UK such as the Kent Weald, Queniborough, Armthorpe, Lympstone, Adswood, etc. which all adjoin farmland whose soils are derived from the bunter/triassic sandstone and greensand formations - all notorious Mn deficient (43) soils where local fanners have professed to use 'copious amounts' of liquid Mn sprays and sewage sludge when consulted.
However, soils from fields adjoining the victims' homes in both Queniborough and Armthorpe were analysed for extractable Mn and averaged excessive levels of 85 mg/L and 115 mg/L respectively, with vegetation averaging excessive Mn levels of 245 mg/Kg and 577 mg/Kg 212.5mg/Kg respectively (Table 2). These exceedingly high levels of Mn recorded in low Mn soil districts suggests that an industrial (dye, brickworks, etc.), fertiliser atmospheric source of Mn is causing high contamination of these areas.
Furthermore ' the increased combustion of Mn tricarbonyl (MM'I) as a lead replacement in high-octane leadfree fuels (12) will considerably increase atmospheric levels of various toxic Mn oxide byproduct compounds in the environment around highways and the 'take-off end of airport runways, etc., Significant levels of airborne Mn originate from emissions vented from steel, glass, ammunition, paint, dye, oil-refining, brick/ceramic and other Mn-associated manufacturing plants (11,12,35,44). Mn dioxide vapours are also emitted from wearing gas masks and the dry cell batteries housed in radio transponder collars hung around cattle and zoo animals necks in Europe.

CELL CULTURE TRIALS PROVIDE EVIDENCE FOR A Mn SUBSTITUION AT PrP's Cu DOMAIN RESULTING IN THE FORMATION OF A PROTEASE RESISTANT PrP ISOFORM
Brown et al. have repeatedly demonstrated that PrPc is a metalloprotein which preferentially binds to Cu in vivo (15) and how its expression protects the cell against challenge by oxidative stress (45). This same team later demonstrated that PrPc exerts a SOD-like antioxidant activity once Cu is conjugated to the octapeptide repeat domain on PrP (6,46). Brown et al. challenged both recombinant PrP- and PrP-expressing astrocyte cell cultures with Mn (5) using mediums containing optimum Cu or deficient levels of Cu. Mn conjugated to PrP's octapeptide repeat Cu domain transforming PrPc into a protease resistant PrPsc isoform after aging. The resulting Mn-PrPsc accumulated into fibril structures.

PATHOLOGICAL1CLINICAL CORRELATIONS BETWEEN TSEs AND CHRONIC Mn TOXICITY INDICATE AN UNDERLYING DISTURBANCE OF Mn METABOLISM IN TSEs
Arnyloid fibrils have been isolated in post-mortem CNS samples drawn from those who have died of Mn-induced dementia (47) and the progressive neuropsychiatric degenerative 'manganese madness' (locura manganica) syndrome afflicting Mn miners (48). Amyloid fibrils composed of misfolded protease resistant PrP comprise the key neuropathological hallmark of the TSE-diseased CNS (1,2).
Other key neuropathological features consistently observed in TSEs (1,2), such as shrunken and distorted basal ganglia (in the putamen and caudate nucleii), astrogliosis, arnyloid plaques, neuronal loss, atrophy, etc. are also recognized as key distinguishing features of chronic Mn intoxication (47-5 1).
Mn 3+ accumulates almost exclusively in the CNS during contexts of chronic Mn intoxication (9), particularly in the mitochondria of astrocyte cells, the pituitary, the retina, the pineal (9,11,12), etc. where Mn3+ is thought to initiate widespread auto-oxidizing chain reactions (7,9,10). High levels of free Mn in astrocytes (9) provide an ideal opportunity for an in situ ceruloplasmin mediated oxidization of Mn (9,52) to occur (astrocytes have been found to express ceruloplasmin (53)) following exposure to invasive CNS-penetrating environmental oxidants such as lipophilic organophosphate insecticides (54) or xeno-oestrogens (55), which upregulate ceruloplasmin turnover. Mn 3+-mediated autooxidization of catecholamine receptors causes a depletion of serotonin, dopamine, noradrenaline turnover (7,10,56,57) in mammals who have been chronically intoxicated by Mn. Interestingly, the brains of scrapie-affected sheep and CJD-affected humans demonstrate an identical pattern of catecholamine depletion (58-62).
A French team has observed a 10-fold increase in Mn levels and low copper levels in postmortem CNS tissue of CJD victims in relation to levels recorded in CJD-free control tissues (63). The results of this work have been duplicated by a further UK study currently under peer review.
The clinical profile of chronic Mn neuropsychiatric syndrome displays a near-identical sequence and range of symptoms (11,35,47,48,64-66) to those encountered in TSEs (1,2,18,67). Both syndromes demonstrate a brief psychiatric phase which is followed by a fatal and progressive neurodegenerative phase encompassing the following symptoms: depression; insanity; confusion; oculogyric crisis; insomnia; unexplained generalized pain; asthenia; self-neglect; aggressiveness; immaturity; delusions; hallucinations; withdrawal; slurred speech; paranoia; mutism; myoclonic jerks; shuffling or cock walk gait; ataxia; excessive laughter/ childlike grins; cogwheel on wrists; dystonia; aphasia; and neurological disturbances involving pyramidal/extrapyramidal and cerebellar-related effects.

Fig. 4(A) Proposed function of healthy 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.


Fig. 4(B) Proposed dysfunction of pathogenic proxidant Mn 3 + prion, Mn loaded / Cu depleted PrP fails to scavenge / quench charge emitted by hyper-photoexcited xenophotosensitiser and Mn prions; thereby subjecting CNS to a lethal prooxidant melt down.

Table 1 Levels of extractable manganese, copper and zinc in soil / vegetation samples drawn from CJD/Scrapie clusters in Calabria / Sicilly and Sardinia respectively. (Sample collections; 17 October 2000, 15 October 2000 and 16 November 2000). Hamlet x = CJD affected hamlet; Former x = Former homevillage of CJD affected families.

The majority of the biochemical and pathological hallmarks of TSEs indicate an underlying pathogenic process that is pivoted upon oxidative stress as the destructive vehicle. The presence of increased levels of cholesterol/decreased amounts of phospholipids and gangliosides in membranes (1), an abundance of lipofuscin inclusions (68), increases in intraneuronal free calcium (69,70), reduction in NAD diaphorase/monamine oxidase (71,72), increases of citrulline/ornithine in blood sera (73), decrease in membrane fluidity (1), rupturing of lysosomal membranes (72), increases in mitochondrial DNA strand breakage (52), etc.
Mn3+ initiated autooxidation could trigger off chain reactions of lipid peroxidation of CNS membranes accounting for many of the more specific idiosyncratic facets surrounding PrP modification in TSE pathogenesis (see Fig. 4); such as the protracted release of PrP's glycolipid anchor from the cell membrane (75) and the sulphoxidation of the 'infamous' methionine sites on PrP (76) (as encoded by codon 129 in all cases of nvCJD to date (17)) and modification of N-linked oligosaccharide side chains (75,76), plus the aggregation of protease resistant PrP isoforms and other cellular debris into the fibril 'tombstone' features which hallmark the CNS pathology of TSE victims (1,2,75).

DOES PrPc PLAY A FUNCTIONAL ROLE IN NEUTRALIZING THE RADICALS GENERATED BY A RANGE OF ENVIRONMENTAL OXIDANTS?
PrP expression has been consistently upregulated in vitro, in response to oxidative challenge of PrP neuroblastoma cells (45).Loss of PrP's Cu-mediated antioxidant SOD function (6) following an Mn substitution at PrP's Cu vacated domain (5), coupled to a low external availability of antioxidant cofactor minerals Se/Cu/Zn in the environment (4) could impair the overall antioxidant capacity of the CNS to neutralize incoming challenge by environmental oxidants.
PrP mRNA is intensively expressed in various neuroectoden-nal cell lines, such as retinoblastoma/melanoma, and in epithelial cell lines (77). Such a distribution suggests that PrP could putatively serve as a critical 'back-up' link in the porphyrin/melanin chromophore chain as a type of 'photooxidative shock absorber' *
Ultra violet radiation is widely recognized as producing superoxide radicals in the contexts of UV-induced degradation of tryptophan (7 p. 291), following UV absorption by some porphyrin and exogeno-us chemical photosensitizers (dyes, etc.) (7) p. 62) and when LTV is absorbed by the pheomelanin type of melanin chromaphore (7, p. 141/62) which fails to self-neutralize the superoxides generated by their own UV absorption (14). PrP's Cu-mediated antioxidant activity could serve to neutralize these superoxides, as well as singlet 02 and other interchangeable radicals resulting from the impact of incoming oxidants.
However, the distribution of PrP mRNA expression is not exclusively confined to the aforementioned types of ectodermal cell. PrP mRNA can also be expressed - at albeit less intensive concentrations - in lung alveolar, some gastrointestinal membranes, the tonsils, appendix, the olfactory/nasal tract, etc. (77,2), which may imply that our mammalian biological systems have harnessed PrP's antioxidant capacities to scavenge a wide array of incoming oxidative assaults delivered by several types of environmental oxidizing agent (e.g. UV, ozone, metals, pesticides, microwaves (mobile phones, radio transponder collar in bovines) etc.) (7) via several exposure routes (e.g. ocular, skin, lung, gastrointestine tract, nasal--olfactory, etc.). Exposures to ozone, heavy metals, pesticides and microwaves are all recognized to generate superoxide and other radicals in biological systems (7,8, p. 396).
The specific route of exposure and the specific oxidative potency of the environmental oxidant involved, coupled to the specific PrP genotype of the exposed individual, could dictate which 'strain' of TSE will emerge.

EVIDENCE SUGGESTS A METABOLIC INTERRELATIONSHIP EXISTS BETWEEN MN, UV CHROMOPHORES AND PrPc IN THE PHOTOBIOLOGICAL RESPONSE
Mn/PrPc association with UV chromophores
Interestingly, Mn is one of the key metals that are integrated into the molecular structures of UV-absorbing chromophores in both mammals and plants (10,13,11, 12,78,79), hence the industrial use of Mn in luminescent materials. Mn melanins are involved in UV absorption in mammals, where the free radicals that are integral to the structure and function of melanin (14) are employed as electron acceptors in the process that neutralizes radicals generated by UV-mediated photooxidation - the negative side-effects of the healthy photobiological response.
The most intensive concentrations of Mn in the mammal appear in the mitochondria-rich melanocyte cells of the retina, the pineal, the skin and pituitary (11,12) - key regions where Mn chromophores are expressed for absorbing UV photon energy and/or its resulting 'excitation energy' that is relayed in the form of fluorescence emission to further acceptor/donator molecules down the line (8,7); ultimately transferring that energy to influence turn over of the central melatonin-serotonergic- endocrine pathways which preside over the circadian regulation of certain reproductive, sleep/wake, behavioural functions etc. (80).
Intriguingly, PrP mRNA is expressed in these same locations - retino melanoma epithelial, etc. (77) suggesting that a shared functional role may exist between PrP and Mn chromophores; where each constitutes a contiguous link in the metabolic chain that neutralizes singlet oxygen and superoxide types of radical generated by the aforementionined contexts of UV absorption.
PrP and Mn coexist within many specialized cells and mitochondria-rich zones throughout the CNS; such as the hippocampal gyrus, cerebellum, reticular formation, neocortex, astrocyte cells (9,48,85,1,2) - areas which are all implicated in the daylight-darkness circadian melatonin-serotonergic-endocrine pathways (80).
Blind/pinealectomized sparrows and neonatal rats have still maintained normal endocrine response to the external light cycle (86), suggesting the presence of a hitherto unknown capability for extra-ocular/pineal photoreception in mammalian organisms. If light is exclusively introduced directly to the hypothalamus of female rats via quartz rods, the oestrous cycle has still been significantly influenced (87). Light has also been shown to penetrate deep into the brain of mammals that have skulls as thick as sheep (88)!
The co-presence of Mn and PrP in the mitochondria rich CNS astrocytes (9,85), etc. suggests a shared metabolic function or interaction between the two. In this respect, Brown et al. have found that PrP expression influences Mn uptake (5) into cells.

THE PrPc-CHROMOPHORE BOND: DOES Cu-PrPc QUENCH SINGLET 0 / LUMINESENCE GENERATED BY UV EXCITED CHROMOPHORES/XENO-PNOTSENSITISERS?
The additional complicating factor of significant levels of various systemic xeno-photosensitising chemicals that have been consistently observed in TSE ecosystems raises the distinct possibility that the Cu histidine domain of PrPc may perform a specific protective role in bonding to these exogenous photosensitiser pollutants (Fig. 4). For PrPc's Cu domain is known to bond 'in vitro' to the photosensitisers; congo red dye, porphyrins, etc. (81-83), thereby scavenging/quenching the singlet oxygenllight energy that is generated when these molecules are exposed to UV radiation or internal light emissions. This possibility is supported by many trials that have demonstrated the role of Cu as a quencher of singlet O luminnescence - one such study demonstrates how CU2+ inhibited the production of singlet oxygen and chemiluminescence after it had been conjugated onto a photoenergised formamidine photosensitiser pesticide molecule called Amitraz (84).
In this respect, the capacity for 'in vivo' expression of PrPc on the surface of phagocytes (neutrophils, etc.), in microglial cells, the gut wall and lipid membranes (2), may simply indicate an extension of the photo-excito absorbing function of PrPc, where Cu-PrPc is employed to scavenge/quench singlet oxygen and the resulting light emissions (luminescence) that are generated in these various contexts, e.g. by activated phagocytes, the respiratory burst of microglial cells, worm/fungal infestations in the gut and following lipid peroxidation of membranes respectively (7, p387).
The possibility of a metabolic interrelationship existing between PrP and porphyrin/phthalocyanines can be speculated following trials which demonstrated how several types of porphyrin bond to PrP histidine sites and block the conversion of normal PrPc into abnormal PrPsc (83).
The intense concentration of tyrosine in PrP's amino acid sequence (77) may indicate that PrP performs a dual melanin-like role in the photobiological response; e.g. by serving as both a scavenger of the radicals generated by photooxidative stress and - by virtue of PrPs tyrosine amino acids - as a chromophore which quenchs the energy of fluoresence emitted by a prior-in-line donor molecule such as melanin or a xeno-photosensitiser. As the degree of efficiency of fluorescence transfer is totally dependent upon the conformational status of the chromophore molecule involved (89), the putative role that PrP performs as a 'photooxidative shock absorber' may collapse once Mn has substituted at the vacated Cu domain on PrPc causing formation of the misfolded protease-resistant PrP isoform.
Furthermore, the low copper status of the CNS resulting in mammals dependent upon low-Cu ecosystems (85) Cow Cu recorded in TSE ecosystems (4)) would cause a considerably reduced turnover of melanin synthesis due to the sole dependence of melanin's catalytic pathway on the Cu-mediated oxygenase enzyme, tyrosinase (89). The same scenario would apply to the turnover of the Zn dependent chromophore, phthalocyanine (89), whose turnover would decrease in mammals thriving off Zn deficient ecosystems (4).
The low-Cu/-Zn that is characteristic of TSE ecosystems (4) would therefore predispose mammals thriving off such foodchains to an inadequate synthesis of protective chromophores, thereby rendering such individuals highly vulnerable to both the deleterious impact of photooxidative stress and to the loss of the protective effect that these chromophores exert in inhibiting the conversion of PrPc into its rogue prion form (83).

PrPc metabolic link to the circadian cycle
Evidence also exists for a direct functional association between PrPe and the circadian response (91,92). One of the few clinical abnormalities that have been consistently observed in mice engineered for PrP knock-out entails a fundamental disturbance in CNS regulation of the circadian rythmn; thereby indicating some functional/regulatory role for PrPc at some stage of the UV-pineal-melatonin-serotonergic pathway. Interestingly, melatonin levels are regulated by Cu levels, whilst levels of Cu, Mn and melatonin in serum follow the circadian cycle (93).
Retinal involvement in the early clinical/pathological stages of TSE
Retinal degeneration has been commonly described in scrapie, transmissible mink encephalopathy (TME), CWD, CJD, Kuru, BSE (2,94-98) with degeneration chiefly affecting the photoreceptor layer of the retina (99-106). The retinal degeneration coincides with the onset of spongiform, degeneration (101) and invariably appears very early in the course of disease before the development of neurological dysfunction (100,96). Interestingly, extracts from the Mn-rich retina and pituitary of TSEdiseased CNS tissues carry the highest titre of 'infectivity' in transmission trials employing TSE-diseased CNS (1). Furthermore, TSE infectivity of the retina has been demonstrated in retinal tissue extracted prior to the development of visible lesions (102).
The optic nerve also carries TSE infectivity with evidence existing for anterograde (103) and retrograde (104) transport of the prion agent along the optic nerve connecting to the brain. Astrocytosis of the optic nerve head has also been observed in CJD (105).
Ocular surgery (involving the cornea, etc.) and ocular tonometry are considered to be predisposing risk factors for CJD (1,2). Whilst the hypothetical consensus on this risk is based on the reductionist perspective that ocular surgery opens up another route for TSE infection, the true relevance of this association could lie with the fact that the ocular disturbances involved are merely manifesting the primary clinical stages of the TSE syndrome resulting from a collapse in PrP and other antioxidant capacities to neutralize photooxidative stress.
Other clinical disturbances in TSEs that could relate to a breakdown in the photobiological respone, involves a range of visual disturbances in 17% of CJD patients (agnosia, diploplia, blurred/distorted vision, etc.), and pruritis of exposed skin surfaces - particularly prevalent in scrapie, BSE, CWD, etc. (1,2). Cortical blindness in TSEs (1)(2) relates to lesions in the visual cortex.
A study of acute high dose effects of UVB on biological systems demonstrates that a range of cancer causing lesions can be induced in DNA: formation of dimer compounds, crosslinking, product additions to bases, chain breaks, etc. (106). Whilst these lesions have seemingly never been investigated in the ocular pathology of TSEs, the lack of ocular/skin cancers as predisposing risk factors for TSEs could merely indicate that the normal DNA repair systems have successfully suppressed any carcinogenic reactions likely to emerge following the long-term chronic exposures to above average doses of UV
However, Eva Mitrova reports that a high percentage of CJD sufferers in the Slovak clusters had been previously afflicted with retinal pigmentosa - a condition where UV induced DNA lesions in the retina fail to repair because of an inborn recessive defect in the expression of repair enzymes in these genotypes. However, rnitochondrial DNA strand breaks (74) and crosslinked proteins (1,2) have been identified in the fibril tombstones of scrapie affected CNS tissue.

SPATIAL EPIDEMIOLOGICAL CORRELATIONS BETWEEN AREAS OF HIGH LEVEL UV EXPOSURE AND HIGH INCIDENCE FOCI OF SPORADIC/FAMILIAL TSEs
One hitherto unexplained geographical characteristic common to the location of sporadic TSE clusters involves the isolated rural nature of the TSE-aftected communities and their position at high altitudes on peaked volcanic, preCambrian, mountain ranges that remain snow-covered for the majority of the year, examples being the CJD clusters in the High Tatra mountains of Slovakia (27), the Calabrian Aspromonte mountains (adjoining Mount Etna) (34), the Kofu Mountain in japan (102), the Highlands of Papua New Guinea (18), CWD cluster in deer of the Rocky Mountains in Colorado (21,22), scrapie clusters in sheep of the N. Icelandic mountains (32,33), the Aragon mountains in Spain (108), the Brecon Beacons in the UK, and more recently the Sopramonte mountains in Sardinia (109).
A correlation exists between these high-incidence TSE cluster mountainous localities and the areas where acid rainfall is prevalent (26,110). Acid rain unlocks the availability of Mn and other cations in such foodchains.
It is also widely recognized that the chronic hypoxia of high-altitude living renders mammals more susceptible to oxidative stress (111) as well as increasing the permeability of their blood/brain barriers to cations such as manganese (112).
It is also widely recognized that these high-altitude environments are naturally challenged by higher levels of UVA radiation as well as the more potent UVB wavelength radiation (110, 113, 106) which, in turn, generates high ground levels of tropospheric ozone gas in the more polluted atmospheres (110, 113-116). Both UV and ozone invoke oxidative stress in biological systems (7).
Photokeratitis or 'snowblindness' traditionally develops in those who live/work in the snowclad mountain environs as a result of visual contact with the higher intensities of UV photons reflected from the snow (114). Higher UV intensities are also encountered in coastal locations where sand and sea reflect the UV rays (106,114). One excellent example of a putative association between this phenomenon and TSE incidence can be found in the Calabrian hamlet where 20 cases of CJD have erupted since 1995 - 20 years since they were moved from their remote mountaintop village and rehoused by a government-funded scheme in a new coastal settlement. A combination of their newly constructed white concrete houses/streets (unique to this area), widespread coastal view and the surrounding bare white sandstone hillside terraces (planted with young olive trees) has caused the development of a 'UV hotspot' that is unprecedented in the area.
UV radiation is also more intense in the broad vicinity around volcanoes where chlorinated emissions have thinned the ozone column in the stratosphere above - thus permitting greater intensities of UV to temporally penetrate the Earth's surface following erruption (110, 114). Rural communities also receive significantly greater intensities of UV radiation than urban communities. This is due to the high intensity of pollutants emitted from urban areas into the troposphere above - such as nitrogen and sulphur dioxides - which serve to scatter and absorb incoming UV radiation before the rays reach the ground (106,113,114).
Whilst the highest international incidence rate of scrapie in Icelandic sheep (32,33) could be partly attributed to the high Mn/low Cu recorded there (4), the protracted 23-hour daylight interval of the Arctic summer may fulfil the further photooxidative prerequisite required for initiating TSE. Whilst the lower elevation of the sun's rays in Iceland, (being diminished near the Arctic pole), serves to decrease the overall summertime intensity of UV exposure in that region (114), this would not entirely compensate for the photooxidative impact of a prolonged 23 hours of UV exposure encountered during clear weather conditions - the ozone layer being thinner above Iceland than at the equator. Furthermore, clinical signs of scrapie are usually first recognized in August, at the end of the summer mountain-grazing period (32).
An occupational risk category for CJD has always centred around people who spend a greater part of their occupational and/or leisure time outdoors in rural areas (1,27-29,34, 107, 117, 121) often at higher altitudes or on the coast; e.g. those most exposed to UV such as farmers, foresters, Naval/RAF personnel, horse-riders, pet-keepers, milkmen, builders, market-gardeners, keep-fit fanatics and, more recently in respect of nvCJD, young Western European people who generally aspire to the tanned image and are sufficiently economically privileged to spend increased amounts of leisure time holidaying abroad/sunbathing on the beach or exposing themselves to recently introduced artificial sources of UV via trendy laser lighting/solaria/sunbeds (122-125,126). However, significant incidences of CJD also arise in those employed in various indoor occupations, such as caterers, nurses, dentists, laboratory workers, plumbers, hairdressers, cleaners, metal cutters, water treatment workers, etc. (119-12 1), and, incerestingly, all of these professions are cited as occupations associated with potential risk of overexposure to UV (106) - where UV may be employed artificially in flytrap strip lighting used in catering/food processing premises, or as UV germicides in water/sewage treatment, hospitals/dentists, public lavatories, laboratories and schools, or as used in arc welding, hairdressing salons, glass blowing, plasma torches, tobacco irradiation chemical analysis, pharmaceutical laboratories, projector lamps, ink curing lamps, drying lacquers or resins in photocopying/printing, etc. (106,116). Phototherapy with UV is also employed to treat babies/young mammals for vitamin D deficiencies or jaundice derived from hyperbilirubaemia or kernicterus (126).
Other mammalian species affected by TSEs involve wild or domesticated animals such as deer/sheep/ goats/cats/cows/zoo animals (2) who are often compelled or simply choose to spend a greater part of their daylight hours feeding/resting in unshaded open countryside. The grant-supported trend of removing woodland and hedgerow from agricultural land in the UK during the 1970s and 1980s left farm livestock with little choice other than to graze pastures where access to shade was unavailable, thus exacerbating any UV-related problems.

THE PRESENCE OF PHOTOSENSITIZING CHEMICALS IN TSE CLUSTER FOOD CHAINS
Concurrent exposure to high intensities of UV and foodstuffs/synthetic chemicals containing systemic photosensitizing chemicals has also been observed in the TSE cluster zones. These substances bond to chromophores, such as melanin, forming long-term stable complexes which impair the photoabsorption process, thereby exacerbating the prooxidant side-effects of UV by generating singlet oxygen and/or superoxide radical (8) (7, p. 62).
This phenomenon is well illustrated by the Greek-Italian community in the Calabria CJD cluster zone, who harvest and ingest relatively large amounts of the locally grown 'bergamot orange' which contains the potent photosensitizer, furocournarin (106).
The increased feeding of significant tonnages of citrus wastes (containing the peel fraction) to UK dairy cows since the early 1980s (38) - a practise likewise adopted by all of the other European countries affected with BSE - would have exposed these herds to furocournarins: on a daily basis.
Furthermore, the deer and sheep in Colorado's/ Slovakia's TSE cluster zones consume large quantities of the alfalfa legume (22,4) which can contain potent levels of the photosensitizer 'psoralen' during damp conditions (126). Various 'terpenes' contained in pine needles and in the aromatic atmospheres around pine forests are potent photosensitizers which are ingested by the deer and human populations involved in the Colorado and Slovakian TSE cluster zones respectively.
Another TSE-affected species, the nocturnal mink (1, 2), can recieve a combined treatment of psoralens and UV for enhancing the pigmentation of their pelts prior to slaughter.
Icelandic sheep are recognised to encounter problems with ingestion of the photosensitiser plant, asphodel, particularly in the wetter valleys where scrapie is more prevalent.
The close upwind proximity of oil refineries and plastic factories to the recently emerged scrapie foci in Sardinia and Sicily, and of a former dye factory in the village of Queniborough and Adswood, which host nvCJD clusters and all circumstantially suggest that environmental exposure to the chemical photosensitizers found in these products(106) may play a contributory role in the aetiology of TSEs (Fig.4).

DOES THE RECENT INCREASE IN COMBINED EXPOSURE TO A COCKTAIL OF OXIDIZING AGENTS (ULTRAVIOLET A+B /OZONE /SYSTEMIC INSECTICIDES) AND Mn POLLUTANTS RELATE TO THE RECENT ERUPTION OF nv TSEs IN N.EUROPE? (SEE FIG. 3).
Interestingly, exposure to UV radiation of the UVA wavelength (above 320 nm) and more potent UVB wavelength (290-320 nm) is significantly increasing as a result of the thinning of the stratospheric ozone layer (115). Mammals have not as yet evolved to cope with exposure to UVB rays which can potentially cause severe photobiological damage to the skin, retina and CNS (113,127) because of its higher energy per photon value (114). The initial mechanism of damage hinges on the chromophore molecule generating reactive oxygen intermediates as a consequence of absorbing UV radiation.
Whilst ozone depletion has apparently occurred over most of the Earth's surface since the late 1970s, the problem is particularly prevalent around the unpopulated polar regions (114). However, ozone depletion is becoming increasingly severe over the populated mid-latitude regions of Northern Europe, where a 4.3% decrease of column ozone over the period 1979-1995 was recorded (113). The decrease varies seasonally, with a threefold greater loss occurring from December to April each year as a result of various pertinent prerequisites simultaneously coming into play - cold conditions, winter sunlight, greater solar elevation and an increase of stratospheric pollutants have all combined to catalyse the destruction of stratospheric ozone (114). Ozone levels and UV intensities can be unpredictable; they can vary dramatically throughout the course of a single day (12 7).
The extremely cold winters of 1992 and 1993 recorded unusually low ozone levels over Northern Europe, with record low levels recorded at Lerwick and Camborne in the UK (113).
Intriguingly, the onset of the clinical stages of both nvCJD and BSE predominantly occurs during this winter/early spring period (120,121,128), whilst the UK epidemic of BSE reached its peak incidence rate in 1992 (12 8), putatively suggesting that any bout of intense UV sunlight exposure encountered during this low stratospheric ozone winter period could be responsible for initiating an in situ oxidization of the dormant Mn2+-PrP conjugate in the retina into its Mn3+/Mn4+-PrP potent autooxidizing form.
The fact that the UK, followed by some other N.European countries, is the foremost culprit in the unrestricted emission of anti-ozone precursor chemicals on kg of chemical emitted per hectare of total land area basis (26,110,114) perhaps explains some of the reasons underlying the increasing severity of UV penetration in the N.European mid-latitude region of the northern hemisphere (113, 114).
The more astute foresight of authorities presiding over other mid-latitude northern countries such as the USA, Canada, Norway and Sweden would appear to have paid off. For instance, these countries endeavoured to control emissions of chlorine-and bromine-containing halocarbons by banning CFC propellants back in the late 1970s. They also compelled use of catalytic converters to curb ozone-depleting motor car exhaust emissions, whilst also taking a guarded approach towards the development of supersonic aircraft technology (110,114) - where the World Meteorlogical Organization's Report on the Scientific Assessment of Ozone Depletion 1994 Executive Summary warned that atmospheric loading of nitric oxides, etc. in the stratosphere of the North Atlantic flight corridor, etc. would lead to significant reductions of ozone colurrin levels in the northern hemisphere.
Whilst levels of UV are likewise increasing in Australasia, etc. in the southern hemisphere, lower levels of Mn pollutants and higher levels of Cu in most of their populated ecosystems could have helped preserve their PrP function and its ability to scavenge the radicals generated by environmental oxidants.
In consideration of ethnic variations in constitutional factors and sun-exposure customs, coupled with the increasing levels of stratospheric ozone as one travels towards the equator - with the correspondingly lower intensities of UVA=B photons recorded on the ground - might explain why the UV-induced malignant melanoma is higher in Northern Europe than in the Mediterranean (129), where the indigeneous population is better acclimatized to UV.
Approximately 50% of cases of nvCJD to date have so far erupted in remote rural and/or coastal communities of the UK where UV radiation/ ground ozone levels are significantly more intense (113, 114). For instance, the SE England cluster of nvCJID in Kent is in the area which experiences the UK's highest sunlight hours (130) and the linear belt of high nvCJD incidence running between Glasgow and Edinburgh (121) is in the area where the incidence of UV-induced melanomas rose by 82% between 1979 and 1989 respectively (131). UVintense coastal areas such as the UK's Channel Islands have hosted the highest incidence rate of BSE in the world (2).
Increased exposure to systemic insecticide oxidants
The contributory role of the systemic organo-dithiophosphate insecticides as a potent in vivo 'oxidizing agent' in the aetiology of BSE has been fully expounded in previous publications (132,133,4). These insecticides were compulsorily used on UK bovines during the 1980s and 1990s at an exclusively high 2 x annual application rate of 20 mg/kg bodyweight of a 20% concentrated oil solution (133). The systemic acting lipophilic liquid was poured along the spinal column/base of the head as a means of exterminating the warble fly parasite which lived inside the cow.
Other types of systemic acting insecticidal oxidants were also simultaneously applied for controlling worms, lice, mange, etc. BSE has been shown to emerge in the UK in zones which were compulsorily treated with warblecide chemicals approximately 4-7 years previously (3). A French study has reported the same delayed lag period existing between the first emergence of BSE and treatment with systemic warblecides (13 4).
The two free sulphur atoms of the dithiophosphate molecule is widely recognized as chelating Cu (135) into a mercaptide ring, thereby creating an artificially induced form of Cu deficiency in the CNS of treated animals. Other Cu-chelating chemicals such as cuprizone are widely recognized as inducing spongiform encephalopathy (13 6) in laboratory conditions.
Furthermore, these insecticides and their in vivo oxone metabolites are widely recognized as generating a high level of oxidative stress in treated animals (137-142), perhaps explaining why PrP expression was upregulated 10 fold after neuroblastoma cells were exposed to 2 p.pm. and 10 p.p.m. doses of a dithiophosphate insecticide (143). These insecticides are also recognized as upregulating ceruloplasmin (54), which leads to an increase in ceruloplasmin-mediated oxidization of metals, such as Mn (52), in the liver or at the CNS astrocytes. Furthermore, if the aetiology of nvTSE turns out to have a hitherto unrecognized association with combined exposures to UV, ozone, systemic insecticides, bovine radio transponder collars or other CNS-penetrating oxidants, then the high intensities of UVA and UVB in mid-latitude N.Europe, plus the UKs exclusive high dose use of systemic warblecide insecticides (significantly lower annual doses were used in France/Eire/Switzerland, Italy, Holland, Spain (133) where BSE incidence was correspondingly lower (128)) may explain why BSE and nv CJD have erupted as an exclusive epidemic in Northern Europe (12 8) to date.

UV-Mn INTERACTIONS AND THEIR PUTATIVE INVOLVEMENT IN THE INITIATION OF TSE PATHOGENESIS
UV photon excitation of Mn could influence biological systems via two avenues of interaction: either as a UVmediated in situ. photoexcitation of 'endogenous' Mn in the retina/skin chromophores, or as a photoexcitation of an 'exogenous'airbom source of Mn particulate which is directly absorbed into the CNS as a short-lived highly reactive species via the nasal-olfactory route (36) or via the lungs. Metals such as titanium, nickel, aluminium, manganese and chlorine (7,116) absorb UV radiation and subsequently adopt a short-lived excited state which can readily interact with molecules in biological systems (113,114); thereby energizing these molecules so that they can generate chain reactions of hpid peroxidation of membranes with formation of hydroxyl radicals, increased intracellular Ca uptake (113), etc. whereupon long-term irreversible damage to proteins, lipids and membranes ensues.
An example of this toxic scenario is well illustrated by the severe health problems encountered by those who have chronically inhaled airborne metals that have been energized into excited states by the UV emitted from the arcs of precision welders working with certain types of metallic medium (116) - such as manganese steel (35). Another toxic occupational scenario involves the use of UV as an ink-drying catalyst in photocopiers/printers, etc., particularly those utilizing inks containing an Mn additive for its rapid hardening/drying property (44). The initial contact between 'energized' Mn and external biological membranes could cause a disruption of the successful incorporation of Mn into Mn-dependent enzyme systems. For successful binding hinges on the availability of the correct Mn species that is complementary to the specificity of the enzyme's active site (9,144). In this respect, once a 'UV-hyperexcited' Mn species is inhaled and makes contact with key Mn-dependent SOD enzyme systems in the lung alveoli, the redox status of this Mn-dependent active site could be critically disturbed; resulting in a greatly diminished activity of Mn SOD in these regions (9). Given that Mn SOD is the radical scavenger enzyme implicated in neutralizing the oxidizing effects of airborne contaminants such as ozone (7) - prevalent in high UV areas - loss of activity would lead to a collapse in the body's first line of defence against these airborne oxidizing agents.
Likewise, UV-hyperexcited Mn could impair the pathway of Mn heme assembly (78) by disrupting the assembly of the final protoporphyrinogen ix-heme product at the end of the Heme biosynthetic pathway where Mn is inserted into the protoporphyrinogen 1X ring inside the mitochondria (89, p. 1016). This bears major relevance to TSE pathogenesis in that porphyrin 1X and phthalocyanines have been shown to protect PrP cells against formation of the protease-resistant PrP isoform (83) associated with all TSEs. Thus, once Mn porphyrin assembly is disrupted, protection against rogue prion formation is lost.
Porphyrins could be inhibiting rogue prion formation (83) (e.g. blocking the transformation of Mn2+PrP into Mn3+PrP) by harnessing their capacity to scavenge free electrons onto the 'acceptor' metals within their pyrole rings, or by scavenging excesses of free Mn/other transition metals within the cell by incorporating them into their pyrole rings during the final stages of porphyrin assembly in mitochondria - thereby serving as an oxidative sink and a potentially useful pharmaceutical for TSEs.

CONCLUSION
This PrP-porphyrin link (83) suggests that normal PrPc may well perform some associative functional role with melanin/porphyrin chromophores by acting as a 'photocitooxidative sink'; where Mn melanins can donate their photoinduced free electrons onto PrP-Cu acceptors; both molecules serving as contiguous links in a relay chain which quenches the radicals generated by the energy of photoexcitation. In this respect, the Cu domain of PrPc may perform a protective role by conjugating with systemic xeno-photosensitiser pollutants, thereby neutralising the singlet oxygen and chernilunimescence that is generated by the photoenergization of these molecules (Fig. 4)
It is concluded that TSE pathogenesis is initiated once Mn has substituted at the vacant Cii domain of PrP (15, 16) in the CNS of susceptibile mammals who reside in high-Mn/low-Cu environs (4). PrP subsequently looses its Cu-mediated antioxidant function (6), thereby imparing PrP's capacity to neutralize singlet oxygen and super-oxide radicals generated by UV-excited melanin A porphyrin/xeno-photosensitiser chromophores. Mn 2+ is subsequently oxidized into a potent Mn3+/~+ autooxidizing species and full-blown TSE pathogenesis ensues.
TSE outbreaks are currently escalating across many regions of N. Europe, presenting a potentially serious public health crisis. The reasons for these outbreaks cannot be explained by the conventional hypothesis. For instance, BSE outbreaks have erupted for the first time in Germany, Spain and Italy, while continuing to increase in European countries already affected such as France, Ireland and Portugal (128) despite bans on meat and bone meal inclusions into their cattle feed rations being implemented back in 1990. A situation has developed where a larger proportion of these countries' total BSE cases involve cattle that were born after their respective bans on meat and bone meal.
This disturbing trend - coupled with the recent rapid increase of many other strains of TSE (scrapie (128, 109), CJD, nvCJD (12 1)) across European countries - raises the urgent need for govemment-backed research programmes to reopen truly independent investigations which aim to unravel the causal riddle of TSEs. Preventative measures and pharmacological therapies for controlling TSEs can only be effectively implemented once the original cause of this insidious disease has been thoroughly comprehended.

ACKNOWLEDGEMENTS
Thanks to Nigel Purdey (London), Dr David Brown (Cambridge), Professor Michel Bounias (Avignon), Professor Sigurdur Sigurdarson (Keldur), Professor Maurizio Pocchiari (Rome), Professor Bob Will (Edinburgh), Dr Eva Mitrova (Bratislava), V1adimira Popikova (Svidnik), Dr Ciriaco Ligios (Sassari), Dr Giuseppe Ru (Torino) for discussion and assitance.

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