CA2342085A1 - Sterol glucoside toxins - Google Patents

Abstract

In one aspect, the present invention discloses the neuronal excitotoxicicity of sterol glycosides. In alternative embodiments, sterol glycosides that are characterized by neuronal excitotoxicicity are .beta.-sitosterol-.beta.-D-glucoside (BSSG) and cholesterol glucoside.

Description

STEROL GLUCOSIDE TOXINS
FIELD OF THE INVENTION
The invention relates to the identification of sterol glucoside toxins, and provides methods for detoxifying the compounds.
BACKGROUND OF THE INVENTION
Sterols are a diverse group of lipids, many of which are found in appreciable quantities in animal and vegetal tissues. Sterols may include one or more of a variety of molecules belonging to C27-C30 crystalline alcohols, having a common general structure based on the cyclopentanoperhydrophenanthrene ring (also called sterane). In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol. There are a variety of other naturally-occurring animal sterols, such as lanosterol (a C30 compound) and 7-dehydrocholesterol, which are illustrative of the structural similarities of sterols. The nomenaclature of sterols is based on the numbering of the carbons as exemplified below for cholesterol:
Sterols are also found in plants. The denomination "phytosterol" has been used for sterols of vegetal origin. Chemically, plant sterols generally have the same basic structure as cholesterol, with differences occurring for example in the lateral chain on carbon 17.
Cholesterol may itself be found in plants. Representative phytosterols are compounds having 29 or 30 carbon atoms, such as campesterol, stigmasterol and beta-sitosterol (stigmasta-5-en-3beta-ol).

Steryl glycosides are sterol derivatives in which a carbohydrate unit is linked to the hydroxyl group of a sterol molecule. In plants, steryl glycosides have been found in which the sterol moiety is composed of various sterols: campesterol, stigmasterol, sitosterol, brassicasterol and dihydrositosterol. Similarly, the carbohydrate moiety may be composed of a variety of sugars, such as glucose, xylose or arabinose. Sterol glycosides may be obtained from biological sources such as plant tissues by a variety of methods (see for example Sugawara et al. Lipids 1999, 34, 1231; Ueno, et al. U.S. Patent No. 4,235,992 issued November 25, 1980). An exemplary plant sterol glycoside is beta-sitostrol-beta-D-glucoside (5-cholesten-24b-ethyl-3b-ol-D-glucoside), for which the formula is give below (also showing the structure of the acylated compound):

O
HO~H
OH
R = H or C15H31C0 Acylated sterol glycosides may be formed in plants when a fatty acid is acylated at the primary alcohol group of the carbohydrate unit (such as glucose or galactose) in the steryl glycoside molecule (see Lepage, J Lipid Res 1964, 5, 587). For example, the 6'-palmitoyl-beta-D-glucoside of beta-sitosterol is reportedly present in potato tubers and the 6'-linoleoyl-beta-D-glucoside of beta-sitosterol is reportedly found in soybean extracts.
Acylated steryl glucoside may be present at relatively high concentrations in a variety of vegetable parts, with the acylated form being generally more abundant that the non acylated sterol glycoside itself (Sugawara et al., Lipids 1999, 34, 1231 ).
Sterol glycosides also occur in bacteria. Helicobacter has for example been described as being particularly rich in cholesterol glucosides (Hague et al., J.
Bacteriol 1995, 177:
5334; Hague et al., April 1996, J Bacteriol; 178(7):2065-70). A cholesterol diglucoside has been reported to occur in Acholeplasma axanthum (Mayberry et al., Biochim Biophys Acta 1983, 752, 434).

Sterols and sterol glycosides have been reported to have a wide spectrum of biological activities in animals and humans (Pegel, et al., U.S. Patent No. 4,254,111 issued March 3, 1981; Pegel et al., U.S. Patent No. 4,260,603 issued April 7, 1981) and techniques for transdermal administration of these compounds have been suggested (Walker, et al. U.S.
Patent No. 5,128,324 issued July 7, 1992). It has been suggested that some plant sterols, their fatty acid esters and glucosides may be useful for treating cancers (Eugster, et al., U.S. Patent No. 5,270,041, December 14, 1993). There have been indications that sterols and sterol glycosides are generally non-toxic, or toxic only at relatively high doses while being beneficial at lower doses (Pegel, U.S. Patent No. 4,188,379 issued February 12, 1980). Some phytosterols are thought to have therapeutic effects, such as anti-tumor properties. Beta-sitosterol is catogorized in the Merk Index, Tenth Edition, as an antihyperlipoproteinemic. It has been suggested that beta-sitosterol (BSS), and its glucoside (BSSG) enhance the in vitro proliferative response of T-cells (Bouic et al., Int J Immunopharmacol 1996 Dec; 18(12):693-700), may have other stimulatory effects as immunomodulators (Bouic et al., Int J Sports Med 1999 May;20(4):258-62), and may therefore be therapeutically beneficial in a wide variety of diseases because of these immunostimulatory properties (Bouic and Lamprecht, Altern Med Rev 1999 Jun;4(3):170-7; Bouic et al., U.S. Patent No. 5,486,510, January 23, 1996).
Cholesterol glucoside (5-cholesten-3b-ol-3b-D-glucoside) is reportedly made by human cells in culture in conjunction with a heat shock response (Kunimoto et al., Jan 2000, Cell Stress Chaperones;5(1):3-7). Cholesteryl glucoside has also been reported to occur in Candida bogoriensis (Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov 7;620(2):322-5).
Sterol glucosides may be hydrolyzed in acid, such as in methanolic HCl (Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov 7;620(2):322-5). Enzymatic cleavage of the beta-glycosidic linkage may also be accomplished, for example by a beta-d-glucosidase. A
thermostable beta-d-glucosidase from Thermoascus aurantiacus that hydrolysed aryl and alkyl beta-d-glucosides has for example recently been reported (Parry et al., 1 January 2001, Biochem J, 353(Pt 1):l 17-127). A steryl-beta-glucosidase (EC 3.2.1.104; CAS
Registration No. 69494-88-8; cholesteryl-beta-D-glucoside glucohydrolase) has been identified from Sinapis alba seedlings that reportedly acts on glucosides of cholesterol and sitosterol, but not on some related sterols such as coprostanol, to hydrolyse the glucoside -producing sterol and D-glucose (Kalinowska and Wojciechowski, 1978, Phytochemistry 17: 1533-1537).
Selective neuronal cell death is the common hallmark of various neurodegenerative disorders. At least two mechanisms of neuronal death have been identified within the mammalian central nervous system: necrosis and apoptosis. Necrosis is generally characterized as a passive form of 'accidental' cell death that follows physical damage and is distinguished by membrane permeability changes leading to swelling of cell organelles and rupture of the plasma membrane (Simonian and Coyle, 1996). In contrast, apoptosis is generally characterized as an active form of programmed cell death involving individual cells that often remain surrounded by healthy neighbors. Apoptosis reportedly requires ATP and protein synthesis (Earnshaw, 1995) and has been characterized by cell shrinkage, membrane blebbing, and genomic fragmentation (Elks et al., 1991; Nagata, 1997).
Both necrosis and apoptosis may be induced by stimulation of neurons by glutamate agonists acting through various glutamatergic excitatory amino acid (EAA) receptor subtypes (Choi, 1995). The actions of glutamate have been classified as either "excitotoxicity" or "excitotoxicity-independent". Excitotoxicity is thought to involve the over-activation of target EAA receptors leading to increased ionic flux. Two main types of excitotoxicity have been described: (1) chronic/slow excitotoxicity, which is thought to result from defects in energy metabolism leading to persistent receptor activation by ambient glutamate (Zeevalk and Nicklas, 1990); and, (2) acute/fast excitotoxicity, which is thought to occur following exposure to high levels of glutamate or glutamate agonists. For example, the over-stimulation of NMDA receptors by glutamate or NMDA may result in increased calcium flux, which in turn may lead to activation of cellular proteases and the activation of other potentially harmful molecules or pathways. It has been suggested that such actions may underlie the damage caused by ischaemia and hypoxia (Choi, 1995; Meldrum and Garthwaite, 1990) or head trauma (Katayama et al., 1988).
Excitotoxicity-independent mechanisms of cell death have been shown to arise due to the accumulation of reactive oxygen species (ROS), elevation of calcium, and the loss of intracellular glutathione (GSH) (Tirosh et al., 2000). Each of these events may induce oxidative stress, described as an imbalance between oxidants (ROS) and antioxidants (GSH, GSH peroxidase, vitamins C and E, catalase, SOD, etc.) with the oxidants becoming dominant (Sies, 1991). Oxidative stress may trigger cellular necrosis (Wullner et al., 1999) as well as apoptosis (Zaman and Ratan, 1998; Hockenbery et al., 1993; Higuchi and Matsukawa, 1999; Nicole et al. 1998) and often arises due to factors leading to GSH
depletion. For a number of reasons, neurons are thought to be particularly susceptible to oxidative stress, and oxidative stress-induced cell death has figured in a number of hypotheses concerning neurodegenerative diseases (see Evans, 1993; Simonian and Coyle, 1996; Palmer, 1999; Russel et al., 1999) and aging (Verarucci et al., 1999).
Toxins present in the environment may play a role in human pathology. For example, agenized wheat flour was the most common source of processed flour in much of the Western world for the first fifty years of the 20'h Century (see Shaw and Bains, 1998;
Campbell et al., 1950) and was later found to contain methionine sulfoximine (MSO) in high concentration.
MSO induced epileptic seizures in experimental animals ((Newell et al., 1947), an action that was not understood but thought to arise due to MSO acting to inhibit the synthesis of both GSH and glutamine (Meister and Tate, 1976). Subsequent studies have revealed that MSO
also has neuro-excitotoxic actions, apparently via NMDA receptor activation (Shaw et al., 1999).
The etiology of various age-related neurological diseases remains largely unknown.
Sporadic forms of Alzheimer's, Parkinson's, and Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS) have been linked to environmental factors that cause neuronal cell death by either by excitotoxicity or by inducing oxidative stress. The experimental and clinical literature has been taken to support a potential role for excitotoxins in some forms of neurodegeneration, notably Lou Gehrig's disease and Alzheimer's disease. In particular, abnormalities in glutamate handling/transport have been linked to ALS
(Rothstein et al., 1990, 1992, 1995) and domoic acid, a kainate receptor agonist, has been shown to be causal to memory losses much like those reported in Alzheimer's disease (Pert et al., 1990).
Oxidative stress has also been linked to the same diseases, particularly following GSH
depletion (see Bains and Shaw, 1997). Excitotoxicity and oxidative stress may in fact be innately linked in that neural excitation, particularly over-excitation which occurs in excitoxicity, may generate free radicals acting to increased oxidative stress (Bindokas et al., 1998).

The following abbreviations may be used in the present application: ALS, amyotrophic lateral sclerosis; ALS-PDC, ALS-parkinsonism dementia complex;
AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ATP, adenosine triphosphate; BSSG, (3-sitosterol-(3-D-glucoside; EAA, excitatory amino acid; GIuR, glutamate receptor; GSH, glutathione; LDH, lactate dehydrogenase; MSO, methionine sulfoximine; NMDA, N-methyl-D-aspartate; ROS, reactive oxygen species; SOD, superoxide dismutase.
SUMMARY OF THE INVENTION
In one aspect, the present invention discloses the neuronal excitotoxicicity of sterol glycosides. In alternative embodiments, sterol glycosides that are characterized by neuronal excitotoxicity are (3-sitosterol-(3-D-glucoside (BSSG) and cholesterol glucoside.
In one aspect of the invention, BSSG is identified as a toxin present in the seed of the cycad palm (Cycas circinalis), historically a staple of the diet of the Chamorro people of Guam. Cycad seed consumption has been linked to ALS-parkinsonism dementia complex (ALS-PDC), an endemic neurological disorder of Guam (Kurland, 1988).
Accordingly, in various embodiments, the present invention provides methods of treating foods to reduce the concentration of sterol glycosides such as BSSG in the foods. In some embodiments, the foods to be treated may for example include plant materials.
An alternative aspect of the present invention is the demonstration that mice fed cycad flour containing BSSG have severe behavioral abnormalities of motor and cognitive function, as well as significant levels of neurodegeneration in the cortex, hippocampus, spinal cord, substantia nigra and other CNS regions measured post mortem. Accordingly, in one aspect the present invention provides an animal model for studying neurodegenerative disease, in which a non-human mammal is fed an excitatory neurotixic sterol glycoside such as BSSG.
In one aspect, the present invention demonstrates that BSSG may mediate neuronal glutamate release followed by NMDA receptor activation. Accordingly, in one aspect the present invention provides in vitro assays for modulators of cytotoxic action, such as assays for identifying compounds that interfere with cytotoxic neuronal glutamate release mediated by BSSG. Lactate dehydrogenase assays may for example be used to assay cell death in vitro in conjunction with administration of BSSG and putative inhibitors of cytotoxicity.
In an alternative aspect, the invention provides kits for detecting BSSG, for example to detect BSSG in foods.
In an alternative aspect, the present invention discloses the toxicity of cholesterol glucoside.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Shows neuro-excitotoxic action of cycad extract (7 x washed cycad chips) demonstrated by in vitro indices of cycad-induced neural activity and toxicity on rat cortical slices. A.
Cortical wedge recording of adult rat neocortex. Drugs were administered to the medium bathing each wedge by gravity flow and neural activity differentially recorded as a field potential. MK801 (MK) blocked the NMDA and cycad-induced depolarizations as did AP5 (not shown); NBQX
blocked only the AMPA response, but had no effect on the cycad response (not shown). B. Cortical slice assays for LDH release following exposure to various compounds. Cycad fractions in the same concentration as applied to induce depolarization gave greater LDH release than that evoked by NMDA. The effects of both were attenuated by APS. Mg2+ diminished LDH release while freeze-thawing slices maximized cell death. *P<0.05, Student's t test.Drug concentrations: NMDA (N), 20~M; AMPA (A), 10~M; cycad: 1: 50 dilution of crude extract of washed cycad in Krebs-Heinseleit buffer (Cyc).
Figure 2. Actions of isolated BSSG fractions on rat cortical slices. A. Field potential recording of isolated cycad sterol glucoside fraction D-2 (15 pM) compared to NMDA (20 pM), other plant sterol glucosides (ouabain or emicymarin, 50 ~M), the (3-sitosterol aglycone (10 ~M), or D-2 plus APS (10 ~M). Arrows indicate onset of drug application.
B. LDH
release following exposure to the same BSSG D-2 fraction (75 ~M) compared to NMDA (50 ~M), the sitosterol aglycone, [3-SS (75 pM); various compounds in the presence of APS (20 pM). Statistics as in Fig. 1.
Figure 3: [3H]-glutamate release in rat cortical slices. A. [3H]-glutamate release with isolated BSSG D-1-2 fraction (25 ~M) compared to NMDA (SO~M) with or without APS (20 ~M). For this experiment, calcium concentration was either 0 (L) or 2mM (H).
Note the calcium dependence for both NMDA and BSSG. B. [3H]-glutamate release by D-2 BSSG
fraction. Concentrations as in B.
Figure 4: Behavioral test results in the mouse model of neurodegenerative disease. A.
Leg extension: the mouse is held by its tail, and in a normal mouse, both of its legs flex out (a score of 2 is recorded). If one or both of the legs do not flex out a score of 1 or 0 is given accordingly. B. Gait Length: the mouse walks through a tunnel with paint on its backpaws.
Distance between subsequent paw prints is recorded as the gait length (stride length). C.
Rotarod: the mouse is placed in a rotating cylinder, at increasing speeds. The time to fall of the cylinder and number of spins (rotations with out falling off) are recorded. D. Wire Hang:
the mouse is placed up side down on a wire mesh and time to fall into a padded box is recorded. E. Water Maze: the mouse is placed in a small swimming pool of water and swims to find a hidden platform located near the middle of the pool. Time to find the platform and percentage of time in each quadrant of the pool is recorded. F. Radial Arm Maze: the mouse is placed in a 8 arm maze, in which 4 of the arms are baited with food. Errors are recorded as entries into unbaited tubes and re-entry in to tubes already visited.
DETAILED DESCRIPTION OF THE INVENTION
Animals In vitro experiments were performed on adult (>70do) male Sprague-Dawley colony rats maintained on a light-dark cycle (12 hr:l2 hr). In vivo experiments were conduced using CD-1 colony reared 5-7 mo. old male mice.
Chemicals MSO was obtained from Sigma-Aldrich Canada Ltd. (Mississauga, Ontario). AMPA, NMDA, APS, and NBQX were obtained from Precision Biochemicals Inc. (Vancouver, British Columbia). [3H] CGP 39653 and [3H] glutamate were purchased from NEN/
Mendel Scientific Co.(Guelph, Ontario). LDH kits and DNAase were obtained from Sigma (St.
Louis). TLJNEL kits were purchased from Intergen (ApopTag).(Oxford). Other chemicals were of analytical grade available from BDH Inc. (Vancouver, British Columbia).

Cycad Extracts and Purification of BSSG
Initial experiments were performed with crude cycad flour extracts made by extensively grinding chips of cycad in a small volume of distilled water.
These cycad chips had been extensively soaked over a period of 7 days. This cycad extract was diluted by various factors in Krebs-Henseleit buffer for use in bath application to field potential or LDH
assays. Based on early experiments (e.g., see Fig. 2), cycad fractions were extensively screened for potency based on the size of the evoked field potential response or on amount of LDH released. From each stage, the most potent batch was selected and further separated by column chromatography. The fractions ultimately yielded several variants of a sterol glucoside, (3-sitosterol-(3-D glucoside (BSSG) with a range of molecular weights ranging from 574-576). These fractions have been given fraction identification codes indicating stage in the isolation procedure and are described in the following as D-2, D-1-1, and D-2.
Electrophysiolo~y: Field Potential Recordin~_s Cortical 'wedges' were prepared as described previously (Shaw et al., 1996).
In brief, animals were anesthetized with COZ, decapitated, and a cortical block rapidly removed and placed in cold Krebs-Henseleit buffer containing (in mM): NaCI 124, KC1 3.3, NaHC03 25, glucose 10, KHZP04 1.2, CaCl2 2.4, and MgS04 1.2, bubbled with 5% C'.OZ/ 95%
O2, pH 7.4.
The cortical block was sectioned into 500 ~M thick coronal slices using a Vibratome (Campden Instruments) and the slices cut into pie shaped wedges in which the white matter formed the narrow edge of the wedge. Each wedge was placed on a net across a grease gap between two fluid filled chambers. The cortical side of the wedge was bathed (at room temperature, approx. 25°C) in buffer lacking Mg2+; the callosal portion was bathed in buffer containing Mg2+ to minimize neural activity. Field potentials were differentially recorded between the two chambers using two Ag/AgCI electrodes. Recordings from up to 6 wedges, each in individual chambers, could be made simultaneously for each experiment.
The wedges were continuously perfused on the cortical side with oxygenated, Mg2+-free buffer using a gravity feed system. Using this system, drugs could be rapidly substituted for control media to examine response characteristics. Wedges typically survived for up to 8 hrs.
Responses were recorded on LabViewTM after amplification and A/D conversion and the traces were charted in ExcelTM for WindowsTM. Statistical analysis of peak response amplitude was performed by one-way ANOVA using Bonferroni's post test with GraphPad PrismTM.
f3H]-Glutamate Release Studies Brain slices were taken from cortical blocks in which all subcortical tissue had been removed.
400~M slices were cut using a modified slice cutter (Van Huizen et al., 1989).
Slices were rinsed twice for 5 min in Mg+Z containing Krebs-Henseleit buffer pH 7.4. Incubation media consisted of 100 pM cold glutamate, 20~M APS and 10 ~M DNQX, the latter NMDA or AMPA
antagonists, respectively. 10 nM of [3H]-glutamate was added to the mixture and incubated for lhr at 37°C under in oxygenated atmosphere (OZ/COZ=95/5%). Experimental treatments were performed in 500 1~M
Mg+2 free buffer placed in tissue culture wells containing different concentrations of MSO or isolated BSSG fractions of cycad flour. Slices were removed at the end of incubation period and the supernatant removed for scintillation counting. The supernatant fractions were placed in scintillation vials containing NEN Formula 989 for a minimum of I2 hrs before being counted in a Beckman LS6000 scintillation counter. Results were normalized to the dpm counts of respective controls.
LDH Assays _7_ Cortical slices were prepared as described above in the glutamate release experiments and placed in tissue culture wells containing Krebs-Heinsleight buffer supplemented with 0.0004% H202 and lmg/mL glucose. Extensive previous studies have demonstrated that this medium supports cellular activity for prolonged periods (Van Huizen et al., 1989; Shaw et al., 1996). (Note that hydrogen peroxide, added as the source of molecular oxygen, was not deleterious at this low concentration (see Van Huizen et al., 1989). In our preliminary experiments, hydrogen peroxide did not affect LDH release up to a 1 mM concentration (0.0034%)). All slices were washed twice with buffer for 20 min each at room temperature before incubation in media containing the test compounds for lhr at 37° C. Test compounds included MSO, NMDA, kainate, different concentrations of cycad extract or different fractions or concentrations of isolated BSSG. MSO, NMDA, and cycad/BSSG were each tested alone or in combination with APS, and compared to control slices maintained in buffer alone. For additional comparison and to establish the limits of the method, some slices were freeze-thawed to kill all the cells. Alternatively, some slices were incubated in buffer containing 1.2 mM Mg2+ in order to diminish spontaneous neural activity. At the end of the lhr incubation period, 3 samples (1001 of buffer, each sample) were taken from each well. LDH assays were performed on these samples using a LDH diagnostic kit (Sigma) following the manufacturer's protocol with some modifications. In brief, 0.5 ml of pyruvate solution was mixed with 0.5 mg pre-weighed NADH. 100 ~l of slice medium (free of slices) was added to the mixture and incubated for 30 min at 37°C. 0.5 ml of Sigma coloring reagent (2, 4-dinitrophenylhydrazine in HCI, 2mg/ml) was added to develop the color and the mixture was incubated for 20 min at room temperature. 5 ml of 0.4 N NaOH were added to each tube. After 5 min, optical density was read at 440nm. Standard curves were prepared for each assay using different concentrations of pyruvate solution (0-960 units). LDH activity (in International Units) was calculated from the standard curve and normalized by total protein content of each slice as determined by the Lowry protein assay (Peterson, 1979). One International Unit represents the amount of enzyme required to convert 1 ~mol of substrate/minute at room temperature.
In situ labeling of DNA Fra~mentation/Apoptosis Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-digoxigenin (DIG) nickend labeling (TUNEL) was carried out using an Intergen ApopTag Plus peroxidase kit using the manufacturer's protocol adapted from Gavrieli et al. ( 1992) with some modifications. More specific antibody labels for apoptosis, eg. caspase 3 also showed cell death in the same regions. Briefly, 20 ~M thick coronal sections were cut on a cryostat then fixed in 1 %
paraformaldehyde at room temperature for 2 days. The endogenous peroxidase was quenched by 3% hydrogen peroxide in phosphate buffered solution (PBS). After rinsing with PBS, the sections were then exposed to 11~,L/cmz working strength of TdT enzyme for lhr at 37°C. After washing in PBS, 15~L/cm2 of anti-digoxigenin-peroxidase was applied for 30 min in a humidified chamber at room temperature. Colour was developed by adding 125 ~l DAB substrate working solution for 6 min.
Slides were counter-stained with methyl green for 25 minutes at room temperature. Positive apoptosis controls were generated by pre-incubating sections with DNAase (Sigma). These methods have been successfully used to indicate apoptotic neurons when used in other preparations (Simpson et al., 2000).
Data for LDH and glutamate release experiements were analyzed for significance by one way ANOVA using Dunnett's and Bonferroni's post tests with GraphPad PrismTM.
Results MSO and cycad mechanisms of action in CNS
_g_ MSO, crude cycad extract, and BSSG isolated from cycad seed flour were tested for neural action and neuro-excitotoxicity in a series of bioassays. Figure 1 a shows the neural response to MSO measured as field potential in the cortical wedge preparation from adult rat.
Bath application of MSO led to a relatively rapid depolarizing field potentials over a range of concentrations beginning at approx. 50 ~M. The responses to glutamate receptor agonists NMDA and AMPA are also shown in the traces of Fig. 1 a for comparison. MSO
responses, like those of NMDA, could be blocked by the co-application of NMDA receptor antagonists APS, kynurenate, or MK 801 (the latter not shown here). MSO responses were not blocked by application of AMPA antagonists NBQX or other AMPA antagonists. Figure 1b shows LDH assays for rat cortical slices following exposure to MSO and other excitotoxins. Both NMDA and MSO increased cell death as measured by LDH release, and both treatments were blocked by the addition of APS.
The actions of cycad flour extracts on the cortical wedge preparation are shown in Fig. 2. Cycad extracts gave depolarizing field potentials that could be blocked by MK801 (Fig. 2a) or APS (data not shown), but not NBQX (data not shown). LDH assays confirmed that cycad extract could induce cell death, an effect that was blocked by APS
(Fig. 2b).
Extensive screening of washed cycad extracts led to the isolation of the most neurally-active and toxic compound contained in washed cycad flour. This molecule has been identified as a plant sterol glucoside, (3-sitosterol-(3-D-glucoside (BSSG) (Shaw et al., 1999b; Khabazian et al., 2000). Figure 3 shows results from the cortical wedge preparation (Fig.
3a) and in LDH
assays (Fig. 3b) using isolated BSSG. The isolated BSSG fractions gave similar field potential responses that were blocked by NMDA antagonists. Cell death in LDH
assays was also blocked by NMDA antagonists.
The above data suggested the possibility that both MSO and cycad/BSSG were acting as agonists at the NMDA receptor. We tested this hypothesis using single cell recording and receptor binding methods. Single cell recording in rat hippocampal and cortical cultures did not show any direct action of these molecules on membrane potentials or neural activity.
Similarly, competition binding studies using the NMDA receptor antagonist [3H]-showed little or no competition for binding.
To test whether the actions of MSO and BSSG might act indirectly by releasing glutamate from intracellular compartments, we examined radiolabeled glutamate release from rat cortical slices. Preloaded [3H]-glutamate release was significantly increased in the presence of MSO and BSSG fractions D-1-2 and D-2 (Figs. 4abc) in a calcium dependent manner, and these effects could be blocked by APS.
Initial behavioral studies of mice fed cycad/BSSG have now been performed and will be reported in detail in a future publication (Wilson et al., in preparation).
Cycad-fed animals showed significant and progressive deficits in both motor and cognitive function. Post-sacrifice histological examinations of the brains of cycad-fed animals revealed the presence of significant levels of apoptosis in hippocampal formation, cortex, and spinal cord compared to control mice. Rats fed MSO also showed evidence of apoptosis in CNS.
References Bains, J.S. and Shaw, C.A. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res.Rev. 1997, 335-358.

Bindokas, V.P., Lee, C.C., Colmers, W.F., and Miller, R.J. Changes in mitochondrial function resulting from synaptic activity in rat hippocampal slice. J.
Neurosci. 1998, 18:
4570-4587.
Campbell, P.N., Work, T.S., and Mellanby, E. Isolation of crystalline toxic factor from agenized wheat flour. Nature. 1950, 165: 345-346.
Choi, D. W., Calcium: still center-stage in hypoxic-ischemic neuronal death.
Trends Neurosci. 1995, 18: 58-60.
Cooper, A.J.L. Role of astrocytes in maintaining cerebral glutathione homeostasis and in protecting the brain against xenobiotics and oxidative stress. In:
Glutathione in the Nervous System, Shaw, C.A. (ed.), Taylor and Francis Pub., Washington, 1998, pp. 91-116.
Earnshaw, W.C. Apoptosis: lessons from in vitro systems. Trends Cell Biol.
1995, 5:
217-220.
Ellis, R.E., Yuan, J., and Horvitz, H.R. Mechanisms and functions of cell death. Ann.
Rev. Cell Biol. 1991, 7: 663-698.
Evans, P.H. Free radicals in brain metabolism and pathology. Br. Med. Bull.
1993, 49: 577-587.
Gavreili, Y., Sherman, Y., and Ben-Sasson, S.A. Identification of programmed cell death via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 1992, 119: 493-501.
Higuchi, Y. and Matsukawa, S. Glutathione depletion induces giant DNA and high molecular weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells. Arch. Biochem. Biophys.
1999, 363: 33 42.
Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Millian, C.L., and Korsmeyer, S.J.
Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993, 75: 241-251.
Janaky, R., Ogita, K., Pasqualotto, B.A., Bains, J.S., Oja, S.S., Yoneda, Y., and Shaw, C.A. Glutathione and signal transduction in the mammalian CNS. J. Neurochem.
1999, 73:
889-902.
Katayama, R., Cheun, M.K., Gorman, L., Tamura, T., and Becker, D.P. Increase in extracellular glutamate and associated massive ionic fluxes following concussive brain injury. Soc. Neurosci. Abstr. 1988, 14: 1154.
Khabazian, L, Pelech, S.L., Williams, D.E., Andersen, R.J., Craig, U.-K., Krieger, C., and Shaw, C.A. Mechanisms of action of sitosterol glucoside in mammalian CNS.
Soc.
Neurosci. Abstr. 2000, 26: 2074.

Kurland, L.T. Amyotrophic lateral sclerosis and Parkinson's disease complex on Guam linked to an environmental toxin. Trends Neurosci. 1988, 11: 51-53.
Meldrum, B. and Garthwaite, J. Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 1990, L 1: 379-386.
Meister, A. and Tate, S.S. Glutathione and related gamma glutamyl compounds:
biosynthesis and utilization. Annu. Rev. Biochem. 1976, 45: 559-604.
Nagata, S. Apoptosis by death factor. Cell. 1997, 88: 355-365.
Newell, G.W., Erickson, T.C., Gilson, W.E., Gershoff, S.N., and Elvehjem, C.A.
Role of "agenized" flour in the production of running fits. J. Am. Med Assoc. 1947, 135: 760-763.
Nicole, A., Santiard-Baron, D., Cellballos-Picot, I. Direct evidence for GSH
as mediator of apoptosis in normal cell death. Biomed. Pharmacother. 1998, 52:
349-355.
Palmer, A.M. The activity of pentose phosphate pathway is increased in response to oxidative stress in Alzheimer's disease. J. Neural Trans. 1999, 106: 317-328.
Perl, T.M., Bedard, L., Kosatsky, T., Hockin, J.C., Todd, E.C.D., and Remis, R.S. An outbreak of toxic encephalopathy caused by eating muscles contaiminated with domoic acid.
N. Eng. J. Med. 1990, 322: 1775-1780.
Peterson, G.L. Review of the Folin phenol protein quantification method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 1979, 83: 201-220.
Pow, D.V., Barnett, N.L., and Penfold, P. Are neuronal glutamate transporters relevant in retinal glutamate homeostatic? Neurochem Intl. 2000, 37: 191-198.
Rechcigl, M. Rates and kinetics of catalase synthesis and destruction in rats fed cycad and cycasin in vivo. Fed. Proc. 1964, 23: 1376-1377.
Rechcigl, M. and Laqueur, G.L. Carcinogen-mediated alteration of the rate of enzyme synthesis and degradation. Enzym. Biol. Clin. 1968, 9: 276-286.
Rothstein, J.D., Martin, L.J., Kuncl, R.W., Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N. Eng. J. Med. 1992, 326:
1464-1468.
Rothstein, J.D., Tsai, G., and Kuncl, R.W., Clawson, L., Cornblath, D.R., Drachman, D.B., Pestronk, A., Staunch, B.L., and Coyle, J.T. Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis. Ann. Neurol. 1990, 28: 18-25.
Rothstein, J.D., Van Kammen, M., Levey, A., Martin, L.J., and Kuncl, R.W.
Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis.
Ann. Neurol. 1995, 38: 73-84.
Russel, R.L., Siedelak, S.L., Raina, A.K., Bautista, J.M., Smith, M.A., and Perry, G.
Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer's disease. Arch.
Biochem. Biophys.
1999, 370: 236-239.
Shaw, C.A. and Bains, J.S. Did consumption of flour treated by the agene process contribute to the incidence of neurological disease? Med. Hyp. 1998, 51: 477-481.
Shaw, C.A., Bains, J.S., Pasqualotto, B.A., Curry, K. Methionine sulfoximine shows excitotoxic actions in rat cortical slices. Can. J. Physiol. Pharmacol. 1999a, 77: 871-877.
Shaw, C.A., Bains, J.S., Williams, D.E., Andersen, R.J., Pasqualotto, B.A., Cheung, J., Tjandrawidjaja, M., Wilkinson, M., Janaky, R., Craig, U.-K. Identification of a novel excitotoxin from cycad seed: implications for neuronal disorders. Soc.
Neurosci. Abstr.
1999b, 25: 1304.
Shaw, C.A., Pasqualotto, B.A., and Curry, K. Glutathione-induced sodium currents in neocortex. Neuroreport. 1996, 7: 1149-1152.
Shaw, P.J. and Ince, P.G. Glutamate, excitotoxicity and amyotrophic lateral sclerosis.
J. Neurol. 1997, 244 (Suppl. 2): S3-514.
Sies, H. (Ed.). Oxidative stress: Oxidants and Antioxidants, Academic Press, New York, 1991.
Simpson, R.J., Khabazian, L, Williams, D.E., Andersen, R.J., Craig, U., and Shaw, C.A. Apoptotic and non-apoptotic cell death following MSO and cycad treatments. Soc.
Neurosci. Abstr. 2000, 26: 261.
Simonian, N.A. and Coyle, J. T. Oxidative stress in neurodegenerative diseases. Ann.
Rev. Pharmacol. Toxicol. 1996, 36: 83-106.
Triosh, O., Sen, C.K., Roy, S., Packer, L. Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death. Neurosci. 2000, 97: 537-541.

Van Huizen, F., Shaw, C., Wilkinson, M., and Cynader, M. Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability. Mol. Brain Res. 1989, 5: 59 69.
Verarucci, D., Verarucci, V., Vallese, A., Battila, L., Casado, A., De la Torre, R., and Lopez Fernandez, M.E. Free radicals: important cause of pathologies refer to ageing.
Panmineva Medica. 1999, 41: 335-339.
Watanabe, M. Developmental regulation of ionotropic glutamate receptor gene expression and functional correlations. In: Receptor Dynamics in Neural Development, Shaw, C.A. (ed.), CRC Press, Boca Raton, 1996, pp. 73-89.
Wullner, U., Seyfried, J., Groscurth, P., Beimroth, S., Winter, S., Gleichmann, M., Heneke, M., Loschmann, P., Schutz, J.B., Weller, M., and Klockgether, T.
Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function. Brain Res. 1999, 826: 53-62.
Zaman, K. and Ratan, R.R. Glutathione and the regulation of apoptosis in the nervous system. In:
Glutathione in the Nervous System, Shaw, C.A. (ed.), Taylor and Francis Pub., Washington, 1998, pp. 117-136.
Zeevalk, G.D. and Nicklaus, W.J. Mechanisms underlying initiation of excitotoxicity associated with metabolic inhibition. J. Pharm. Exp. Ther. 1990, 257: 870-878.
CONCLUSION
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Given the overlap in the occurrence of particular sterols in plants, animals and other organisms, the present application refers to all such compounds collectively as sterols. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Claims (7)

1. A method of food processing comprising treating a food product containing a neurotoxic sterol glycoside, comprising subjecting the food to conditions that reduce the concentration of the neurotoxic sterol glycoside.

2. The method of claims 1, wherein the neurotoxic sterol glycoside is selected from the group consisting of BSSG and cholesterol glucoside.

3. The method of claim l, wherein the sterol glycoside is cholesterol glucoside.

4. The method of claim 1, wherein the sterol glycoside is BSSG.

5. The method of any one of claims 1 through 4, wherein the conditions are effective to hydrolyse a glycosidic bond in the sterol glycoside.

6. The method of claim 1, wherein the conditions comprise treating the food product with an enzyme that degrades the neurotoxic sterol glycoside.

7. An animal model of neurodegenerative disease comprising a non-human mammal fed a sufficient amount of a food comprising a neurotoxic sterol glycoside to produce symptoms of neurodegenerative disease.