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OF GANGLIOSIDES IN THE HINDBRAIN OF THE ‘ CHICKEN (GALLUS DOMESTICUS) presented by Dennis Michael Bush has been accepted towards fulfillment of the requirements for __Mas.t£r__degree in AnimaLicLence Major professor [hue March 14, 1991 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution 9 IM LIBRARY M'CMQan State L University at PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE I #—I ”—7 MSU Ie An Affirmative Action/Equal Opportunity Institution emana-pd THE EFFECT OF THE DELAYED NEUROTOXIN DIISOPROPYLFLUOROPHOSPHATE (DFP) ON THE DISTRIBUTION OF GANGLIOSIDES IN THE HINDBRAIN OF THE CHICKEN (QALLHS DQMESTIQHS) by Dennis Michael Bush A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1991 ABSTRACT THE EFFECT OF THE DELAYED NEUROTOXIN DIISOPROPYLFLUOROPHOSPHATE (DFP) ON THE DISTRIBUTION OF GANGLIOSIDES IN THE HINDBRAIN OF THE CHICKEN (QALLHS DQMESTIQUS) by Dennis Michael Bush The mechanism of -action of compounds causing organophosphorus ester—induced delayed neurotoxicity (OPIDN) is still a mystery as is the function of gangliosides in the nervous system. To determine if there was a relationship between the development of OPIDN and the relative proportion of endogenous brain gangliosides, hens were injected subcutaneously with diisopropylfluorophosphate (DFP) at a dose of 1 mg/kg body weight. Birds were sacrificed at time intervals corresponding to different stages of ataxia. DFP was found. to have no effect on the concentrations of protein, total lipid, lipid phosphorus, total cholesterol and ganglioside-bound sialic acid in the chicken hindbrain. The effect of DFP on the relative proportion of the major gangliosides in the hindbrain was also examined. DFP affected the relative proportion of GM4, 603, Gle and Gle within the hindbrain. Results suggest that changes in the ganglioside profile began to occur before the advent of neuronal degeneration. ACKNOWLEDGMENTS I would like to thank the members of my guidance committee, Dr. Steven Bursian, Dr. Charles Sweeley and Dr. Richard Aulerich, for their useful suggestions during the preparation of this manuscript. Special thanks are extended to Ellen Lehning for guiding me through the quagmire of computer programs and statistics. Thanks are extended to fellow graduate students, Doug Weisner and Lyla Melkerson-Watson, for taking time out from their busy schedules to teach me how to use a densitometer. Thanks are also extended to Dr. Duke Tanaka for taking pictures of my chromatography plates, and Sharon Debar for helping me with the freeze evaporator. Additional thanks goito Dr. Bursian and Ellen Lehning for making graduate school enjoyable and rewarding. I would especially like to thank my wife, Christina Bush, for her love and support during the preparation of this manuscript. TABLE OF CONTENTS Page LIST OF TABLES...O...........0............OOOOOOOOOOOOOO v LIST OF FIGURES ..... .....OOOOOOOOOOOOOOOOOO ....... ...... Vii INTRODUCTIONOOOO 00000000000 O OOOOOOOOOOOOOOOOOOOOOOO .0... 1 LITERATURE REVIEW... ...... . ............. . ..... ... ....... Gangliosides.......................................... Discovery....................................... ...... Structure, Classification and Nomenclature............ Isolation and Identification.... ....... ............... Distribution.......................................... 15 Biosynthesis and Degradation.......................... 17 Changes During Brain Development...................... 22 Biological Functions.................................. 24 Effect of Injury on Endogenous Gangliosides........... 29 Organophosphorus Ester-Induced Delayed Neurotoxicity.. 31 General Uses of Organophosphorus Esters............... 31 Toxicokinetics and Metabolism of DFP.................. 33 Clinical Signs of OPIDN................. ...... . ...... . 34 Human Exposure to Delayed Neurotoxins................. 34 Initiation of OPIDN................................... 36 Development of OPIDN.................................. 40 Expression of OPIDN................................... 43 Variability of Response............................... 44 @009.)wa MATERIALS AND METHODS........ ........... ................ 46 Husbandry............................................. 46 Experiment 1 .............. ............. ..... .......... 46 Experiment 2.......................................... 47 Brain Removal......................................... 48 Summary of Tissue Analysis............................ 49 Lipid Extraction and Protein Quantification........... 49 Lipid, Cholesterol and Lipid Phosphorus Quantification 50 DEAE-Sephadex Chromatography and Base Treatment....... 50 Dialysis.............................................. 51 Iatrobead Chromatography.............................. 52 Lipid-Bound sialic Acid Quantification.... ............ 53 Thin Layer Chromatography............................. 54 Statistics............................................ 55 RESULTS............................................ ..... 56 Clinical Assessment of DFP-Treated Birds... ........... 56 Experiment 1.......................................... 56 Experiment 2.......................................... 57 Combined Data From Experiments 1 and 2.. ....... ....... 62 DISCUSSION.............................................. 7l Ganglioside Profile of the Chicken Hindbrain.......... 71 Effect of Parathion on Lipid and Protein Levels....... 71 Effect of DFP on Lipid and Protein Levels............. 72 Effect of DFP on Individual Gangliosides.............. 74 Cellular Location of Ganglioside Changes.............. 76 Gangliosides and Susceptibility to OPIDN... ........... 78 Gangliosides and the Mechanism of 0PIDN............... 79 CONCLUSIONS...’ 000000000000 C0.0.0..........OOOOOOOOOOOOO 81 BIBLIOGRAPHYOO ........ 00...... ...... ......OOOOOOOOOOOOOO 82 iv LIST OF TABLES Table Classification of gangliosides according to the structure of their carbohydrate core .......... . ..... Recovery of gangliosides from monkey brain total lipid extracts using different isolation methods. Values expressed as mean : standard deviation....... Distribution patterns of myelin gangliosides of the brain. Values are expressed as a percentage of total ganglioside sialic acid. Table from Cochran et a1. (1982)....................................... Number of birds in the treatment groups of Experiments 1 and 2.... ....... ...... ..... ... ........ Clinical assessment of adult chickens administered a single subcutaneous dose of DFP (1 mg/kg body weight).........00.000.000.00.......OOOOOOOOOOOOOOOO Concentrations of protein, total lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid per gram wet weight (gww) in the hindbrains of control and DFP-treated chickens at 7, 14 and 21 days post-dosing................ ....... Percent distribution of ganglioside-bound sialic acid in the hindbrains of control and DFP-treated chickens at 7, 14 and 21 days post-dosing........... Concentrations of protein, total lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid per gram wet weight (gww) in the hindbrains of control, DMSO-treated (vehicle control), parathion-treated (negative control), and DFP-treated (test group) chickens at 4 days post- dosing .......... . ...... . ................. . .......... 11 21 48 56 58 59 61 10. Percent distribution of ganglioside-bound sialic acid in the hindbrains of control, DMSO-treated (vehicle control), parathion-treated (negative control) and DFP-treated (test group) chickens at 4 days post-dosing.................................... 63 Percent distribution of ganglioside-bound sialic acids in the hindbrains of control and DFP-treated chickens at 4, 7, 14 and 21 days post-dosing........ 67 vi LIST OF FIGURES ure The structure of the ganglioside GMl (From Rapport, 1981)....0O.....0....0.00......OOOOOO......OOOOOOOOO Chemical structure of selected gangliosides. The nomenclature of Svennerholm (1980) is used. According to this method, M, D, and T indicate 1, 2, and 3 sialic acid residues, respectively (Q and P) would indicate 4 and 5 such residues, respectively). G stands for ganglioside and the letters a and b are used to distinguish between positional isomers of sialic acid. Symbols: circle, glucose; square, galactose; hexagon, N-acetylgalactosamine; filled triangle, sialic acid (From Rapport, 1981).......... Schematic representation of the different routes of ganglioside biosynthesis (Tettamanti, 1988)......... The pathways of ganglioside synthesis. Abbreviations are: Cer, ceramide; Glc, glucose; Gal, galactose; GalNAc, N-Acetylgalactosamine; and SA, sialic acid (From Yu, 1983)..................................... The structure Of DFPOOOOOOIO00.000.000.000...0...... The consequences (2a and 2b) resulting from the binding of certain organophosphorus compounds to NTE (From Johnson, 1982)............................ The aging of NTE following phosphorylation by DFP... Thin-layer chromatogram of ganglioside extracts from the hindbrains of (A) control and DFP-treated chickens at (B) 7, (C) 14 and (D) 21 days post-DFP administration. The bovine standard is represented by (S). The arrows indicate gangliosides which were found to differ significantly from controls......... vii 19 20 32 38 39 63 10. 11. Thin-layer chromatogram of ganglioside extracts from the hindbrains of (A) control, (B) parathion- treated, (C) DMSO-treated and (D) DFP-treated chickens at 4 days post-dosing. The bovine standard is represented by (S)............................... 64 The relative percent distribution of GM4, GMl, GD3, GDla, GTla + GDZ, Gle, Gle and Gle in the hind- brains of control (day 0) and DFP-treated chickens at 7, 14 and 21 days post-DFP administration ........ 66 The relative percent distribution of G03, Gle and Gle in the hindbrains of control chickens (day 0) and DFP-treated chickens at 4, 7, 14 and 21 days post-DFP administration....... ..... ................. 70 viii INTRODUCTION The study of organophosphorus-induced delayed neurotoxicity (OPIDN) began with the work of Maurice Smith and his co-workers (1930) at the National Institutes of Health. They sought the cause of a peculiar form of paralysis which affected thousands of people during the 19205. It is now sixty years after their initial studies and the mechanism of OPIDN is still a mystery. More than 40,000 people have suffered from the paralysis caused by organophosphorus delayed neurotoxins (Abou-Donia and Lapadula, 1990). In 1978, the Environmental Protection Agency published guidelines in the Federal Register for testing organophosphorus compounds to determine their ability to cause OPIDN (U.S. Environmental Protection Agency, 1978). According to Abou-Donia and Lapadula (1990), there were more than 1,000 cases of OPIDN in humans during the 19805. Like the mechanism of delayed neurotoxicity, the function of gangliosides is also a mystery. Since their discovery by Ernst Klenk in the 19305, gangliosides have: been studied intensively; These lipids have received.a Igreat.deal of attention because they have neuronotrophic and neuritogenic characteristics. The effect of delayed 2 neurotoxins on lipid components of nervous tissue was studied frequently during the 19605 and 19705, but ganglioside concentrations were not examined. Few studies have examined the effect of physical or chemical insults on the ganglioside profiles of the nervous system. This is unfortunate because much useful information concerning the function of gangliosides could be gathered from such studies. By knowing how a nerve degenerates after exposure to a toxin, one can learn useful information concerning the mechanism of action of the toxin and possible ways of reducing or' preventing neuronal damage. In the following study, the degeneration caused by an organophosphorus delayed neurotoxin will be used as a model for studying the role of gangliosides in the nervous system. LITERATURE REVIEW GANGLIOSIDES Discovery- Ernst Klenk discovered an unusual lipid during the late 19305 while studying the brains of patients who suffered from Tay-Sachs disease. This lipid, which Klenk called "substance X", was abnormally high in the brains of Tay-Sachs patients. Since Klenk thought "substance X" was concentrated in neuronal cells (ganglienzellen), he gave it the less mysterious name ganglioside (Yu, 1983; Wiegandt, 1985). Structure, classification and nomenclature- It was more than twenty years after Klenk’s discovery that.the structures of the major gangliosides of the mammalian brain were determined. It is now known that gangliosides are sialic acid-containing glycosphingolipids. A ganglioside molecule consists of a hydrophilic sialosyloligosaccharide headgroup attached by a glycosidic bond to a hydrophobic ceramide tail (Figure 1). sialic acid residues are attached either to a galactose of the oligosaccharide chain or to another sialic acid. The ceramide portion, consisting of sphingosine and a fatty acid bound in amide linkage, is ll Illilxeeouzzn 36.2% 6.5638318 UH 7.333626 32.3. 333.9 9823. 2-233.. mango: a506,... co 8263350 mu... _ .33 3 ...... . ... 2.. n so: 9%: x I osmiminz -9. no: -n:.-n3-nx.-oz.inx..3. 3.9.63-9...3A}- e: 9:0: o o o: z z z: -mnniinznozfl n:~-nx~inz.. nxflnznnxfi9.6363536}-n3-nz.-nx. oz 1 1 oz 0 oz 1 . z x 12: on... x o: or or my: _ . n INInII n?- 9: Z->na;_aoc83m3n ona mmLPpho occnfiv «macaw H. 43m mflwcnacwm om ”:6 mmzodmommam 93H Amxoa mmcuoxn. HmmHv. 5 located in the plasma membrane. The sialosyloligosaccharide portion extends out from ‘the membrane surface into the extracellular space and significantly contributes to the negative charge of the cell surface (Ando, 1983). Gangliosides can be classified into four series (gala, neolacto, globo, and ganglio) based on the composition of the oligosaccharide chain (Table 1). The majority of the gangliosides of the .brain belong' to the ganglia-series. Gangliosides may differ from one another in the following ways (Rapport, 1981; Ledeen and Yu, 1982): 1. The number of sugar residues may vary. 2. The number and position of sialic acid residues may vary. 3. Glucosamine may substitute for galactosamine. 4. Fucose residues are sometimes present. 5. Glycolyl may substitute for acetyl groups on the sialic acid. 6. O-acetyl groups are sometimes present. 7. The structure of the ceramide may vary. Gangliosides are normally named using the rules established by the IUPAC-IUB Commission on Biochemical Nomenclature (1977) or by a method developed by Svennerholm (1980). In this paper, the shorter notation of Svennerholm (1980) will be used. Figure 2 shows the chemical structures of some of the more common gangliosides of the central nervous system. Even though more than 70 different gangliosides have been detected in the mammalian brain, certain gangliosides 6 Table 1. Classification of gangliosides according to the structure of their carbohydrate core. Series Structure Gala Gal-Cer Neolacto Gal-GlcNAc-Gal-Glc-Cer Globo GalNAc-Gal-Gal-Glc-Cer Ganglio Gal-GalNAc-Gal-Glc-Cer Gal=Galactose, Cer=Ceramide, Glc=Glucose, GlcNAc=N- Acetylglucosamine and, Ga1NAc=N~Acetylgalactosamine Cerornide 6513 WCeromide . G Ceramide M4 ?—Ceromide Gm Go—(ii—o G Dlo WO- DIb D—O—i—O eromc e GMZ WCeromide G le W . G ceramide [)3 f0 Ceramide Figure 2. Chemical structure of selected gangliosides. The nomenclature of Svennerholm (1980) is used. According to this method, M, D, and T indicate 1, 2, and 3 sialic acid residues respectively (Q and P would indicate 4 and 5 such residues, respectively). G stands for serine Direct _ Glycosylation Req/ NUCLEUS Figure 3. Schematic representation of the different routes ganglioside biosynthesis (Tettamanti, 1988). of 20 Cucmdu ”-0 Col-Cu -—-+C.oI'Cei I I Gk - Cu 1 Col - Glc - Cu I Gel-GI: -Ccr -——--—-———«-O I SA I (G...) Gczh'Ac ~ Gol- Gk -Cor I SA (6...) SA I 5A (503) I Go: NAc - GoI- Gk - Cu I Ciel-Grim! ———---—-+ Gol~Gercr I S'A S A 1A com: ‘GOFGK’COI I (on) SA S'A 5A 1 (Gag) 5‘ (Goa) 1:“ (612) 1 I. Gol- GolNk-ecI-Gk-Cu Got-GalNAt-GoI-Gk-Cu Got-GoINAt-GeI-Gk°¢u .. .;. a. I l (6.0 l 5‘ (Goa) l i: (Gin) Gil-Gem“ -Gol-GJt-Cu Gel-GalNAc-GelickiCu Gel-CoINAc-G'elrck-Cer SA 5“ SA 51A 5" fi‘ 1 (60,.) l 5‘ (cm) 1 i: (c...) Gel-GolNk ~GoI-Glt-Cer Gal-GolNk-GeI-Clc-Cu G I I I ' PI SA SA SA SA 5': (67" ) 5'4 5'1 (60") 4 . The pathways of ganglioside synthesis . Abbreviations are: Cer, ceramide; Glc, glucose: Gal, galactose; GalNAc, N-acetylgalactosamine; and SA, sialic acid (From Yu, 1983). 21 Table 3. Distribution patterns of myelin gangliosides of the brain. Values are expressed as a percentage of total ganglioside sialic acid. Table from Cochran et al. (1982). CHICKEN HUMAN RAT MOUSE GM4 31.8 20.3 4.6 4.7 GM3 2.9 3.5 1.4 0.3 GMZ 2.0 3.2 2.0 3.6 GM1 33.8 31.7 63.7 59.6 GD3 5.8 1.8 3.0 4.2 GDla 11.4 7.8 5.8 8.0 GTla+GD2 - 1.2 2.6 4.8 Gle 5.7 19.5 6.6 8.0 Gle 5.0 8.9 6.2 4.2 Gle 1.6 2.1 4.1 2.6 22 1970). The break—down products from these reactions can be recycled by transferral from the lysosomes to the Golgi apparatus (Tettamanti, 1988) (Figure 2). Some inherited disorders are characterized by an overabundance of a ganglioside due to a deficiency in the activity of one of these enzymes. For example, in GM1 gangliosidosis there is a build-up of GM1 due to a defect in B-galactosidase and in Tay-Sachs disease there is a build-up of GM2 due to a defect in B-acetylhexosaminidase (Ando, 1983). Changes during brain development- During the development of the brain, there are changes in the concentrations of individual gangliosides. These changes have been shown to parallel structural changes. For instance, the levels of GD3 and GM3 increase during proliferation of neuronal and glial precursor cells; Gle increases.during'cell migration and arborization; GDla and Gle increase during the formation of synapses; GM1 and GM4 increase during myelination (Seybold and Rahmann, 1985; Mahadik and Karpiak, 1988: Skaper et al., 1989). There is not a constant increase in ganglioside concentrations in the brain of a chicken as it ages. Dreyfus and co-workers (1975) found that the quantity of gangliosides in the chicken brain increases significantly just prior to hatching and then afterwards increases gradually to the adult concentration. The accumulation of gangliosides after 23 hatching parallels brain growth, but the increase prior to hatching does not. other important changes are occurring during development. For instance, glycosyltransferases and sialyltransferases are highest during early post-hatching development and decrease during the adult stage (Mahadik and Karpiak, 1988). Ganglioside patterns within the brain also change after parturition and hatching. There is an increase in the proportions of GM1 and GDla and a slight decrease in the proportions of Gle and Gle in the brain of a chicken as it ages (Dreyfus et al., 1975). This pattern is not universal since brains of humans (Suzuki, 1965) and rats (Vanier et al., 1971) exhibit changes with age opposite to those observed in the chicken. Certain gangliosides are expressed mainly during an animal's early development. The polysialogangliosides formed by the C pathway are important components in the brain of a young chicken, but are negligible in the adult. This is evidence of phylogenetic recapitulation since there is a decrease in polysialogangliosides during phylogeny to higher order species (Hilbig et al., 1981). The disappearance of the polysialogangliosides may also be related to the transition from a heterothermic to homeothermic state of development. The brain ganglioside profiles of different species of birds have been found.to'differ'depending'on*whether‘their'postnatal development is nidifugous or nidicolous (Seybold and Rahmann, 24 1985). Biological functions- Gangliosides may mediate the response of the membrane to environmental factors by affecting the dynamics and fluidity of the membrane. The relationship between gangliosides and membrane fluidity'is the basis for Rahmann's hypothesis (1983) that gangliosides are involved in synaptic transmission. According' to this hypothesis, calcium ions complex: with gangliosides at the synapse before an action potential occurs. At this time, the gangliosides are clustered together making the membrane rigid and impermeable to the ions. An action potential causes a local change in ion concentration outside the membrane which displaces calcium ions from the gangliosides resulting in ganglioside dispersal. This dispersal increases the fluidity of the membrane resulting in an increased permeability to calcium ions. The resulting influx of calcium triggers the release of a transmitter into the synaptic cleft. Gangliosides may be involved in the transfer of information through the plasma membrane. They may accomplish this function by influencing the production of secondary messengers or the generation of ion fluxes through the plasma membrane. Gangliosides have been found to influence the following membrane-associated proteins: adenylate cyclase, sodium-potassium ATPase, protein kinase, cyclic nucleotide 25 phosphodiesterase and tyrosine kinase (Fishman, 1988). Gangliosides may influence the activities of these enzymes by modifying the microenvironment of the membrane around them (Yates, 1986). Gangliosides may also have an effect on sodium and calcium channels (Spiegel, 1988). Gangliosides may function as receptors in the central nervous system (CNS). Gangliosides have many characteristics that make them ideal receptors. For instance, they are located.in the outer surface of the plasma membrane, they have a variety of structures and they carry a charge (Wiegandt, 1985). They are thought to bind the following ligands: viruses (sendai and influenza), bacteria and bacterial toxins (enterotoxin, cholera, tetanus and botulinum), fibronectin, interferon, neurotransmitters (serotonin), glycoproteins, and calcium ions (Fishman, 1988). Gangliosides themselves may also function as ligands. There is evidence for the existence of a ganglioside-binding protein on the surface of cells. It is thought that when a ganglioside binds to this protein, it alters either the activity of a kinase or ionic flux. Altering kinase activity will in turn alter ‘the rate of protein. phosphorylation (Schengrund, 1990) . Gangliosides have been found to both stimulate and inhibit the phosphorylation of proteins (Goldenring et al., 1985; Chan, 1987). Altering the rate of phosphorylation could affect the ratezof cell growth or affect cellular functions (Schengrund, 1990). 26 The discovery that changes in the levels of gangliosides during development parallel CNS differentiation provided evidence that gangliosides play an important role in neuritogenesis. This hypothesis was supported by Purpura and Baker (1977) who showed that GM1-gangliosidosis caused affected neurons to display meganeurites and other types of aberrant sprouting from regions of the cell that had elevated levels of stored gangliosides. Kasarskis and co-workers (1981) provided additional support for this hypothesis when they produced long-lasting morphological and behavioral abnormalities by administering GM1 antibodies to developing animals. These early studies led many researchers to add gangliosides to cell cultures and administer them to animals with the hope of stimulating neuritogenesis. Exogenously administered gangliosides are able to cross the blood-brain barrier, concentrate in the plasma membranes and function like endogenous gangliosides (Tettamanti, 1988). Exogenous gangliosides were found to enhance neurite formation in a wide variety of cultured cells, including neuroblastoma, pheochromocytoma, dorsal root ganglion and fetal brain of the rat and chick. Gangliosides were also found to accelerate reinnervation in 2119 (Yates, 1986; Gorio, 1986: Sahel, 1988; Mahadik and Karpiak, 1988; Schengrund, 1990). In animals, early post-lesion treatment with GM1 has been shown to improve neuronal cell survival of dapaminergic, 27 serotonergic, and cholinergic neurons (Skaper et al., 1989). The mechanism by which gangliosides produce their neuronotrophic effects is unknown. According to Mahadik and Karpiak (1988), ganglioside5'may’berable tijrevent additional damage following an initial neuronal insult by: (1) enhancing the neuron’s responsiveness to neuronotrophic signals. It is hypothesized that the increase in trophic activity following a brain injury may be insufficient to prevent additional damage to the neuron. In yitrg studies show that GM1 enhances the effect of neuronotrophic factors (NTFs) on NTF-responsive neuronal cells. .Antibodies to GM1 have been shown to block NGF-induced neuritogenesis (Skaper et al., 1988). (2) protecting the neurons from imbalances between excitatory and inhibitory stimulation. Neural injuries are characterized by imbalances in neurotransmitter activities which may be harmful to the neuron. For example, it has been found that excitatory amino acid transmitters have neuronotoxic effects following ischemic brain damage (Mahadik and Karpiak, 1988). GM1 has been found to protect the brain against various biochemical and fUnctional deficits during cerebral ischemia (Skaper et al., 1989). (3) protecting the structure and function of neuronal membranes. Neuronal injury may affect the structure of neuronal membranes by altering their lipid content. Changes in membrane lipids will affect membrane fluidity and stability 28 (Mahadik and Karpiak, 1988) . Exogenously administered GM1 has been found to reduce toluene-induced increases in fluidity of synaptosomal membranes (von Euler et al., 1990). A reduction in membrane lipids may lead to increases of free fatty acids which are thought to be pathogenic. Gangliosides may prevent increases in fatty acids by preventing calcium influx. Preventing the influx of calcium into the cell will prevent the activation of phospholipases which release fatty acids from phospholipids (Mahadik and Karpiak, 1988). Calcium will also not be able to enhance the activities of calcium- dependent proteases and kinases which have been associated with neuronal degeneration (El-Fawal et al., 1990). Neuronal injury may adversely affect the function of plasma membranes. Alterations of membrane lipids and destabilization of the plasma membranes will adversely affect the activities of membrane-bound enzymes like sodium-potassium ATPase and magnesium ATPase. Exogenous GM1 may prevent the loss of enzyme activity by stabilizing the membrane or by interacting with the enzyme directly. The failure of membrane function may also lead to cellular ionic imbalances and.edema. Gangliosides may reduce ionic imbalances by stabilizing the membrane and/or by protecting the activity of an important ion pump like sodium-potassium ATPase (Mahadik and Karpiak, 1988) . 29 Effect of injury on endogenous gangliosides- The heavy metals lead and mercury alter the pattern of gangliosides in the central nervous system. Young rats given a diet containing 1% lead showed an increased proportion of GDla and decreased proportions of Gle and Gle (Stephens and Gerber, 1981). After chronic exposure to mercury, the basal ganglia of monkeys showed an increase in GD2, Gle, Gle and Gle, and a decrease in GM2, GM1 and GDla (Ando, 1983). Following pentylenetetrazol-induced convulsions in rabbits, there was a relative decrease in Gle and Gle, and increases in GDla and GM1 (Kostic, 1981). Rats administered a single pharmacological dose of alcohol had a reduction in the levels of GM1, GM3, GDla, Gle and Gle in their brains (Klemm and Foster, 1986). Few studies have examined the effects of trauma on the synthesis and composition of gangliosides. After Sbaschnig- Agler and co-workers (1984) injected a radiolabeled ganglioside precursor into the crushed eye of a goldfish, there was an eight-fold increase in the amount of radiolabeled ganglioside within the visual pathway. Yates and Thompson (1978) found a 64% increase in the amount of gangliosides within a rabbit sciatic nerve by three weeks after transection. There was an increase in GM2, GM3 and GD3 and a decrease in Gle, Gle and GDla. Seifert and Fink (1983) electrolytically lesioned the entorhinal cortex of a rat which resulted in the partial deaf ferentiation of the dentate gyrus. 30 The lesion stimulated axonal sprouting and synapse formation which resulted in an increase in the biosynthesis of gangliosides, with GD2 having the most notable increase. Ganglioside metabolism is altered by the presence of neural tumors. There is an increase in polysialogangliosides and decrease in monosialogangliosides as a result of increases in cell density. There are also significant changes in ganglioside distribution in the following central nervous system diseases: Creutzfeld-Jacob disease, multiple sclerosis, and amyotrophic lateral sclerosis (Ando, 1983). The central nervous system is not the only location where there are changes in ganglioside profiles following an insult. Bouchon and co-workers (1985) found a reduced proportion of GM3 in pathological thyroids. 31 ORGANOPHOSPHORUS ESTER-INDUCED DELAYED NEUROTOXICITY General uses of organophosphorus esters- There is a high potential for exposure to organophosphorus esters due to their wide range of uses. In industry they are used as plasticizers, gasoline additives, stabilizers in lubricating and hydraulic oils, and as flame retardants. In agriculture, they are used as insecticides, anthelmintics and defoliants. Organophosphorus esters function as biocides by inhibiting acetylcholinesterase. In addition to the effects caused by the inhibition of this enzyme, some organophosphorus esters also cause a delayed neurotoxic effect known as organophosphorus ester-induced delayed neurotoxicity (OPIDN) (Metcalf, 1982). Diisopropylfluorophosphate (DFP) (Figure 5) was first synthesized during World War II by McCombie and Saunders (1946), but due to security reasons their results were not published until after the war. DFP has never been used in warfare, agriculture or industry, but it has been used in research. It. has frequently been used. in cholinesterase inhibition studies because: (1) it is very effective at inactivating acetylcholinesterase and certain other esterases: (2) it has a high lipid solubility which allows it to enter the central nervous system and (3) it is relatively specific (Taylor, 1980). Figure 5. 32 /C33 0 Owen \\p/ \CH CH F/ \o —— CH CH3 The structure of DFP 33 Toxicokinetics and metabolism of DFP- When DFP is administered intravenously at a dose of 0.1 mg/kg body weight, it binds to serum proteins and is distributed to the lungs. It accumulates in the liver and kidney following the administration of larger doses (Abou- Donia, 1984). The penetration of DFP into the brain is slow and dose dependent. DFP appears to bind to the tissues it encounters first with less of the toxin reaching the more isolated tissues like the brain (Ramachandran, 1967). When DFP is injected into a femoral artery, it distributes in a proximal to distal gradient in the sciatic nerve (Howland et al., 1980). Aromatic esters usually undergo metabolic activation to metabolites that are more neurotoxic, whereas aliphatic esters like DFP are direct acting neurotoxins. The liver is the main site of detoxification following intraperitoneal injection of DFP. After being absorbed by the liver, it is bound to microsomal esterases and hydrolyzed to a product more readily excreted. Intravenous injection of DFP into guinea pigs results in the formation of 0,0-diisopropylphosphate. 0,0-diisopropylphosphate is mainly excreted in the urine and to a lesser degree in the bile. Small amounts of DFP are excreted without being broken down. Large toxic doses of DFP result in increased biliary excretion of DFP and its metabolites with reduced urinary excretion (Abou-Donia, 1984). 34 Clinical signs of OPIDN- In humans, the clinical signs of delayed neurotoxicity are observed 8 to 14 days after the initial exposure to the toxin. The first symptoms of OPIDN are tingling, numbness and weakness in the distal portion of the lower limbs. The weakness and ataxia eventually lead to paralysis of the: lower limbs. The upper limbs are also affected in severe cases (Johnson, 1982). Chickens administered an oral dose of 1 ml tri-ortho- cresyl phosphate (TOCP)/kg body weight exhibit symptoms of OPIDN 8-10 days after exposure. At this time there is an unwillingness to walk with a preference for squatting. The birds tire easily and after exertion they walk with a broadening and stumbling gait. (h: the following day the weakness and clumsiness are more pronounced, the feet slap heavily on the floor and the legs are spread widely to maintain balance. During the subsequent.4 or'5 days the legs become progressively more weakened until the bird is unable to stand. During this same period the leg and tail reflexes become progressively reduced until they are virtually non-existent. The wings are sometimes weakened but not to the same degree as the legs (Cavanagh, 1954). Human exposure to delayed neurotoxins— The clinical signs of OPIDN were first noted during the late 19th century in tuberculosis patients who were treated 35 with an uncharacterized mixture of esters called phosphocreosote. OPIDN was not studied in humans until TOCP, a chemical once widely used in industry, was identified as the cause of a peculiar form of paralysis that affected thousands of people in the United States during the 19205. This paralysis was caused by the consumption of an alcoholic extract of Jamaican ginger adulterated with TOCP. "Ginger Jake" paralysis affected approximately 20,000 people during the prohibition era. TOCP was also responsible for the paralysis of 10,000 people in Morocco in 1959 after engine oil containing TOCP was accidentally mixed with cooking oil (Metcalf, 1982). Organophosphorus esters are popular as insecticides because they have a Ihigh acute toxicity, they are not persistent in the environment, and they are relatively inexpensive to manufacture. These characteristics made them logical replacements for organochlorine insecticides. However, organophosphorus esters are not "perfect" insecticides because certain of these compounds have caused delayed neurotoxicity in humans (Baron, 1981). The compounds mipafox and leptophos were the most highly publicized of the OPIDN-causing pesticides. Their synthesis was halted after several workers in manufacturing plants developed delayed neurotoxicity. A total of seven organophosphorus pesticides have been associated with delayed neurotoxicity in humans (Cherniack, 1988). 36 Initiation of OPIDN- The mechanism of organophosphorus ester-induced delayed neurotoxicity is unknown. OPIDN is most likely initiated by the binding of the toxin, or an active metabolite of the toxin, to a protein called neuropathy target esterase (NTE). NTE is a membrane-bound protein found mainly in neuronal tissues whose physiological function is unknown (Johnson, 1975). Dudek and Richardson (1982) found the following NTE activities in various tissues of the adult hen (values are expressed as percentage of brain NTE activity): spinal cord (21%), peripheral nerve (1.7%), gastrocnemius muscle (0%), pectoralis muscle (0%), heart (14%), liver (0%), kidney’ (0%), spleen (70%), spleen lymphocytes (26%), and blood lymphocytes (24%). The NTE activity in the hen brain was determined by Dudek and Richardson (1982) to be 2426 i 104 nmoles/min/gm wet weight (mean :1; S.E.M.). Subcellular fractionation of hen brain homogenates revealed that NTE activity is low or absent in nuclear, mitochondrial, and myelin fractions and enriched in synaptosomal and axonal preparations (Richardson et al., 1979). The NTE activity within a tissue is determined by using one of its substrates. Because no physiological substrates of NTE have been discovered, the synthetic compound phenyl valerate is used. Phenyl valerate is also hydrolyzed by enzymes other than NTE so a differential assay is needed to separate the activity of NTE from the activity of the other 37 enzymes. The activity of NTE is determined by a colorimetric assay which measures the amount of phenol released following the hydrolysis of phenyl valerate. The activity of the enzyme is determined after preincubating the tissue with: (1) paraoxon and (2) paraoxon plus the delayed neurotoxin, mipafox. Paraoxon is used in the assay to saturate all the esterases, other than NTE, which hydrolyze phenyl valerate. By subtracting the enzyme activity found in (2) from that found in (1), it is possible to determine the amount of phenol formed solely from the hydrolysis of phenyl valerate by NTE (Johnson, 1982). Normally, the total NTE activity must be inhibited by 70-80% for OPIDN to occur. Sprague and Bickford (1981) administered repeated subneurotoxic doses of DFP to chickens and produced delayed neurotoxicity in birds by inhibiting the total NTE activity by less than 50%. Carbamates and certain phosphinyl and sulphonyl compounds inhibit NTE by more than 70%, yet they do not cause delayed neurotoxicity. These compounds do not cause OPIDN because, unlike delayed neurotoxins, they are unable to undergo the process of aging. Aging occurs when an "R" group of the phosphorylated protein is cleaved from the phosphorus, leaving behind a negatively charged protein (Figure 6). The aging of NTE following its phosphorylation by DFP is shown in Figure 7. The non-delayed neurotoxins which are capable of binding to NTE do not have hydrolyzable bonds and therefore cannot undergo the process 38 TARGET PROTEIN E0” 6) IN VIVO ORGANOPHOSPHCRYLATION (or PHOSPHINYUTION, 9ch OF ACTIVE SITE OF THE TARGET INHIBITS NTE ACTIVITY. MODIFIED O R n, TARGET o-R PROTEIN R BIOLOGICAL RESPONSES IN VIVO DEFEND ON THE NEXT CHEMICAL CHANGE @ IFIorZ R—P bonds are C-O-P lFBothR-Pbonds are C-P I Phosphate or Phosphonatc) i Phospninatel 'TI-iEN' . _ THEN Aging IS possuble Aging‘ is impossible (Usually rapid on NTE) \ 9’0 NO CHANGE o-P. R N0 NEUROPATHY also {ORIMHW a AGED INHIBITED NTE IS PROTECTED (”mung m AGAINST NEURWATHIC 0.P. ESTERS mmgrgs NEUROPATHY SO ANIMAI. IS PROTECTED AGAINST THEIR NEUROPATHIC EFFECTS. Figure 6. The consequences (2a and 2b) resulting from the binding of certain organophosphorus compounds to NTE (From Johnson, 1982). ‘ 39 Figure 7. The aging of NTE following phosphorylation by diisopropylfluorophosphate. 40 of aging (Johnson, 1982). The R group is thought to be transferred to another site on the NTE molecule (Williams, 1983). The aging of DFP has a half-life: of about 2-4 minutes (Clothier and Johnson, 1979). Development of OPIDN- It is not known how the aging of NTE leads to the formation of lesions in the nervous system. The increased negative charge of the membrane due to the aged NTE or the transfer of the R group may disrupt an important function of the membrane resulting in neuronal damage (Johnson, 1982). It is possible that the aged NTE mimics the activity of another phosphorylated protein. This mimicry could harm the axon because of the slow turnover rate of phosphorylated NTE as compared to other phosphorylated proteins (Richardson, 1984). Bouldin and Cavanagh (1979b) studied the early stages of axonal degeneration in nerve fibers from cats dosed with DFP. They found unique varicosities in nerve fibers before degeneration occurred. These swellings were associated with intra-axonal and/or intra-myelinic vacuoles. They suggested that these vacuoles were due to a breakdown in control mechanisms regulating intracellular and extracellular ionic gradients. A net influx of ions into the axon or the myelin would lead to an influx of extracellular fluid resulting in the development of vacuoles. 41 Studies in the past have examined the effect of delayed neurotoxins on lipid metabolism in the peripheral nervous system. In the sciatic nerve of the hen, there was an increase in cholesterol levels and a decrease in triglyceride content following exposure to TOCP. The levels of phospholipids, diglycerides, cholesterol esters, and proteolipids, as well as the activity of phospholipase were not altered (Morazain and Rosenberg, 1970). The sciatic nerves of chickens exposed to TOCP aerosols had reduced levels of cerebrosides, triglycerides and lysolecithins and increased levels of cholesterol esters, mono- and diglycerides, lecithin and ceramide. There were also changes in the fatty acid compositions of cholesterol esters, sphingomyelin, cerebrosides and phosphatidylserine in the sciatic nerves of the chickens (Berry and Cevallos, 1966). Very few studies have examined the effect of delayed neurotoxins on lipid metabolism in the central nervous system. In cerebral slices from chickens dosed with TOCP, there was no change in the distribution of neutral lipids or phospholipids. There was also no change in whole brain levels of proteolipids (Morazain and Rosenberg, 1970). Mipafox had no effect on whole brain levels of phospholipid, sphingomyelin, cholesterol, cerebroside or total lipids in chickens. In the spinal cord myelin from chickens dosed with sumithion, there was an increase in the concentrations of cholesterol and cholesterol ester and a decrease in the 42 concentrations of cerebroside, sulphatide and gangliosides. Sumithion did not cause a significant change in total lipid or phospholipid concentrations (Nag and Ghosh, 1984). No studies to date have examined the effect of delayed neurotoxins on ganglioside profiles. Other important discoveries have been made that may help to explain the development of OPIDN. Seifert and Casida (1982) have evidence that microtubules and microtubule-associated proteins are the target sites for delayed neurotoxins. EfleFawal and co-workers (1989) found that the calcium channel blocker verapamil modified the effects of the delayed neurotoxin phenyl saligenin phosphate. El-Fawal and. co-workers (1990) also found. an elevation of calcium-activated neutral protease activity in neuronal tissue and muscle during the period between the inhibition of NTE and the development of OPIDN. Moretto and co-workers (1987) found increasing impairment of retrograde transport in the sciatic nerve from days four through seven after exposure to the delayed neurotoxin, dibutyl dichlorovinylphosphate (DBDCVP) . Delayed neurotoxins may also accelerate anterograde axonal transport (Reichert and Abou-Donia, 1980; Carrington et al., 1989). The phosphorylation of numerous brain and spinal cord proteins that are Ca”- and calmodulin-dependent was enhanced after the oral administration of TOCP to chickens (Patton et al., 1985). The primary proteins affected were cytoskeletal 43 proteins (alpha- and beta-tubulin, MAP-2, and neurofilament triplet proteins) that are phosphorylated by Caa/calmodulin kinase II (CaM kinase II). CaM kinase II, instead of NTE, may therefore be the initial target for OPIDN (Abou-Donia and Lapadula, 1990). Expression of OPIDN- Neuronal degeneration occurs in the peripheral nerves, spinal cord and the lower brainstem. In the peripheral nervous system, both the sensory and motor nerves are affected. The long myelinated axons with large diameters are affected most severely (Cavanagh, 1954). The initial degeneration occurs at the distal portion, but not the extreme terminus, of the axons. Degeneration of the myelin occurs secondary tx> the axonal degeneration (Bouldin and Cavanagh, 1979 a, b). In the central nervous system, degeneration occurs in the cervical spinal and medullary tracts. The fasciculus gracilis, dorsal and ventral spinocerebellar tracts, spinal lemniscus, glossopharyngeal and vagus nerve are all affected. Terminal degeneration occurs in the following brainstem nuclei: lateral cervical, gracile-cuneate, external cuneate, inferior olivary nuclei, nucleus tractus solitarius, and the reticular formation. Mossy fiber degeneration occurs in the granular layer of the cerebellar folia I-Vb (Tanaka et al., 1990). In the spinal cord of chickens dosed with TOCP, the 44 lumbar portion of the medial pontine-spinal tract is also affected (Tanaka and Bursian, 1989). Variability of response- There is a difference in species sensitivity to the delayed neurotoxic effects of OPIDN. Sensitive species include the baboon, man, chicken, squirrel monkey, water buffalo, horse, cow, sheep, pig, dog, cat, slow loris, duck, pheasant, turkey, ferret and partridge. The less sensitive or insensitive species include the rat, mouse, rabbit” Iguinea pig, hamster, gerbil and Japanese quail. The adult White Leghorn hen (Gallus domestigus) is used as the test animal for OPIDN because its response to delayed neurotoxicity is similar to that of humans (Metcalf, 1982). There is also an age difference in sensitivity to the delayed neurotoxic effects of compounds which cause OPIDN (Barnes and Denz, 1953; Bondy et al., 1960). Johnson and Barnes (1970) injected chicks at ages up to 49 days with DFP at doses of 2-5 mg/kg body weight without the chicks developing delayed neurotoxicity. The chicks were more susceptible to this dosage at 60-100 days of age and highly susceptible from 100-130 days of age. Kittens also did not develop OPIDN after being administered large doses of TOCP (Taylor, 1967). Children also appear to be less sensitive than adults to OPIDN (Cherniack, 1988). The NTE activity of the chick and rat can be inhibited 45 by more than 70% without either animal exhibiting ataxia. Because the initiation steps of OPIDN are similar in the rat and chicken, the difference in species susceptibility to ataxia is likely due to events which occur after phosphorylation of the enzyme (Johnson, 1975). The difference in susceptibility to OPIDN may be due to neuroanatomical differences between species (Morazain and Rosenberg, 1970; Johnson, 1975; Baron, 1981). It is also possible that young and nonsusceptible animals differ from susceptible animals in their ability to repair the neuronal damage caused by the aged NTE (Richardson, 1984). Richardson (1984) hypothesizes that inhibition and aging of NTE above a critical level should lead to pathogenesis irregardless of species and age. Part of the basis for this hypothesis is Novak and Padillas’ (1986) research showing the similarity of NTE found in the hen and rat. The inhibition of NTE activity in the rat and chicken results in peripheral nerve and spino-cerebellar degeneration in both animals, but only the chicken exhibits ataxia (Veronesi, 1984). Veronesi (1984) found clumps of axonal sprouts in the peripheral nervous system of rats dosed with TOCP. The hypothesis is also supported indirectly by research which shows that young animals have a greater potential for repair than do older animals (Richardson, 1984) . MATERIALS AND METHODS Husbandry- Thirty 85-week-old White Leghorn (EELS W) hens were obtained from the Michigan State University Poultry Science Research and Teaching Center and housed individually in cages measuring 41 cm long x 21 cm wide x 51 cm high in an environmentally controlled room. The hens were provided with a commercial layer ration (Purina Layena) and water ad libitgm and the photoperiod was maintained at 16h light : 8h dark. The temperature and the relative humidity of the room were 21 degrees celsius and 30%, respectively; The birds were allowed to adjust to room conditions at least two weeks prior to the administration of the test chemical. EXPERIMENT l Fourteen hens were assigned randomly to four groups. Group 1 consisted of four hens which were injected subcutaneously over the right breast muscle with di- isopropylfluorophosphate (DFP) (Sigma Chemical Company) at a dose of 1 mg/kg body weight in a volume of 1 ml/kg body weight. Dimethyl sulfoxide (DMSO) was used as the vehicle. The birds were given a 1 ml intraperitoneal injection of 46 47 atropine sulfate (15 mg/kg body weight) immediately prior to, and when necessary after dosing to protect against the acute cholinergic effects of DFP. The birds were killed 4 days after dosing. Just prior to being killed, the degree of ataxia exhibited by the hens was assessed using the 8-point scale of Cavanagh and co-workers (1961). On this scale, 0 indicates no ataxia, 1-2 indicates mild ataxia, 3-4 indicates moderate ataxia, 5-6 indicates severe ataxia, and 7-8 indicates paralysis. The other three groups in this experiment served as controls. Group 2 (four birds) consisted of untreated birds, group 3 (two birds) were vehicle controls and therefore received 1 ml DMSO/kg body weight while group 4 (four birds) were administered by intraperitoneal injection 10 mg parathion [0,0-diethyl-O-(4-nitrophenyl) phosphorothioate] /kg body weight. Parathion was used as the negative control because both DFP and paraoxon (the neuroactive metabolite of parathion) are cholinesterase inhibitors but paraoxon does not cause OPIDN (Soliman et al., 1982). Birds in the parathion and DMSO groups were killed four days after injection. EXPERIMENT 2 Thirteen birds were administered DFP at a dose of 1 mg/kg body weight using the same procedure described for Experiment 1. Four birds were killed on days 7 and 14 post-dosing while five birds were killed 21 days after the administration of 48 DFPu The administration of DFP to each group was staggered so that the birds could be killed on the same day. Three untreated birds were also killed at this time and used as controls. The degree of ataxia was determined for all birds just prior to sacrifice according to the method developed by Cavanagh and co-workers (1961). The number of birds in each of the treatment groups used in the two experiments is shown in Table 4. Table 4. Number of birds in the treatment groups of Experiments 1 and 2. Untreated DMSO Parathion DFP 4D‘ 7D 14D 21D EXP 1 4 2 4 4 EXP 2 3 4 4 5 *Refers to the number of days post-DFP administration. Brain removal- After the birds in the two experiments were killed, their brains were quickly removed and the hindbrain, consisting of the cerebellum and brainstem, was separated from the rest of the brain. The hindbrain was then weighed and frozen for subsequent analysis. The hindbrain was the only region. of the brain assessed because OPIDN-induced 49 degeneration is limited to this area (Tanaka et al., 1990). Summary of tissue analysis- Gangliosides were isolated using a modification of a method described by Ledeen and Yu (1982). At various points in this procedure, aliquots were taken for protein, total cholesterol (cholesterol and cholesterol esters) and lipid phosphorus quantification. Total lipid was measured gravimetrically. Lipid extraction and protein quantification- The hindbrain of each bird was homogenized in 60 mls of chloroform-methanol-water (2:1:0.1) in a 100 ml beaker. Aliquots were taken for protein estimation using the method described by Lowry and co-workers (1951). The beakers containing the homogenates were placed in an oscillating water bath at 37 degrees celsius for 30 minutes. The contents of each beaker were then poured intoIa scintered glass filter (60 mls; 10-15 microns) attached to a 125 ml filtering flask. A vacuum was applied to the flask and the filtrate from each sample was poured into a separate 250 ml Erlenmeyer flask and covered. The pellet remaining in the filter was scraped back into the original beaker and 60 mls of chloroform-methanol— water (1:1:0.1) were added to it. After mixing, the samples were again placed in a water bath for 30 minutes and then filtered. The filtrate from this step was combined with the 50 first filtrate. The above steps were repeated using chloroform-methanol-water (1:2:0.1) and.the filtrate combined with the two previous filtrates. Lipid, cholesterol and lipid phosphorus quantification- The combined filtrates were placed in a previously weighed 250 ml round bottom flaskg The solvent was evaporated from the samples at 40 degrees celsius using a rotary evaporator and the amount of lipid present was measured gravimetricallyu The samples were then dissolved in 10 mls of chloroform-methanol (1:1). Aliquots of the samples were taken for the estimation of total cholesterol using a colorimetric cholesterol kit (Sigma Chemical Company) and for the quantification of lipid phosphorus using Dodge and Phillips’ (1967) modification of ‘the method. developed by' Bartlett (1959) . DEAE-Sephadex chromatography and base treatment- The samples were added to columns containing 4 mls of activated DEAE-Sephadex A-25. The DEAE-Sephadex was activated by placing 2.2 gms of resin in a beaker and mixing it with 30 mls methanol-chloroform-0.8 M aqueous sodium acetate, 60:30:8 (solvent A). After the resin settled, the supernatant was removed by aspiration. This step was repeated three times with fresh solvent A and the resin was allowed to stand overnight.in the same solvent» The supernatant fluid was then 51 removed and the resin was washed three times with 30 ml portions of methanol-chloroform—water, 60:30:8 (solvent B). The resin was made into a slurry by adding 10 mls of solvent Bu The slurry was transferred to a column containing a glass— wool plug. After the resin settled, it was washed with an additional 60-80 mls of solvent B to assure that all the sodium acetate was removed. .After the sample was added.to the column, 10 mls of chloroform-methanol (1:1) were added to elute the neutral and zwitterionic lipids. The acidic fraction was isolated by adding 20 mls of 0.8 M sodium acetate in methanol to each column and collecting the eluates in 250 ml round.bottom flasks“ The acidic fraction was evaporated to dryness at 40 degrees celsius with a rotary evaporator. To eliminate acidic phospholipids (serine phospholipids, inositol phosphoglycerides, etc.) , the samples were dissolved in 20 mls of 0.1 N sodium hydroxide in methanol and left standing overnight. The samples were then neutralized with 1 M acetic acid. The solutions were evaporated to dryness with a rotary evaporator and then placed in a dessicator. A vacuum was applied to the dessicator and run for 30 minutes. After dessication, the samples wereIdissolved.in 10 mls of distilled water. Dialysis- Dialysis tubing was used to remove salts and other small molecular weight contaminants from the samples. The first 52 step of this procedure required tying two knots at one end of a 12" strip of dialysis tubing that had been prewashed in boiling water for one hour. Each sample was added to the open end of a tube with a funnel and pipet. The empty flasks were rinsed two to three times with a small amount of distilled water and the rinsates were added to the tubes. The open end of the tubes were knotted and the dialysis sacs were placed in a 4000 ml beaker of distilled water. The beaker was covered with plastic wrap and placed on a stirrer in a coldroom. The distilled water was changed after 24 hours in order to increase the concentration gradient and thereby allow more contaminants to diffuse from the dialysis tubing into the surrounding fluid. After an additional 24 hours in the coldroom, the samples were poured intoIlzo ml beakers, covered with cheesecloth, and placed in a freeze evaporator for two days. Iatrobead chromatography- After lyophilization, the samples were dissolved in 4 mls of chloroform-methanol (1:1) after which an additional 6 mls of chloroform were added. The samples were added to columns containing pre-washed iatrobeads (Iatron Industries, Inc.). The following method was used to remove contaminants from the iatrobeads: two gms of iatrobeads were placed in a 50 ml beaker. The beads were washed four times with 30 ml portions of methanol-chloroform-2.5 N ammonium hydroxide (60:30:8). 53 The beads were then washed four times with 30 ml portions of methanol-chloroform-water (60:30:8). After the contaminants were removed from the iatrobeads, 10 mls of the methanol- chloroform-water mixture were added to the iatrobeads and the slurry was poured into a column containing a glasswool plug. After the silicic acid settled, it was washed with 20 mls of chloroform—methanol (1:1) and then with 20 mls of chloroform- methanol (95:5). After the sample was added to the column, 25 mls of chloroform-methanol (4:1) were added to elute sulfatides and fatty acids. Ninety mls of chloroform-methanol (1:1) were added to the column to elute the gangliosides. Lipid-bound sialic acid quantification- The solvents were removed from the samples using a rotary evaporator. Each sample was dissolved in approximately 2.5 mls of chloroform-methanol (1:1) and placed in a glass tube with a teflon-lined screw top. The flasks were rinsed with a small volume of chloroform-methanol (1:1) and the rinsates were added to the tubes. The samples were taken to dryness with a nitrogen evaporator and then dissolved in 1 ml of chloroform-methanol (1:1). Aliquots were taken for lipid- bound sialic acid quantification using the procedure described by Svennerholm (1957) and modified by Miettinen and Takki- Lukkainen (1959). The absorbance of the solutions was read at 620 nms with a Staser II Spectrophotometer. 54 Thin layer chromatography- Twenty microliters of each sample were applied as 0.5 cm strips on a 20 by 20 cm silica gel 60 HPTLC plate with a 10 microliter Hamilton syringe. The plate was placed in a paper- lined chamber containing 155 mls of chloroform-methanol-0.5% aqueous calcium chloride (55:45:10). Before being placed into the solution, the plate was allowed to equilibrate in the tank for twenty minutes. The plate was removed from the chamber when the solvent had risen to a point 5 cms from the top of the plate. After the plate was dry, it was sprayed with the resorcinol-RC1 reagent described by Svennerholm (1957). This reagent was made by adding 0.5 mls of 0.1 M copper sulfate to 80 mls of concentrated hydrochloric acid followed by the addition of 10 mls of a 2% resorcinol solution and 9.5 mls distilled water. The reagent was allowed to stand at room temperature for four hours before being'usedn IAfter spraying, the plate was dried with a stream of warm air, covered with a glass plate and heated at 110 degrees celsius for 20 minutes. Gangliosides appeared as purple bands and were identified by comparing them to a bovine standard (ICN Biochemicals) co- chromatographed with the samples. The developed plates were stored in a freezer to prevent the color of the bands from fading. The relative proportion of individual gangliosides within the samples was determined using a scanning densitometer. 55 Statistics- The treatment groups within each experiment were tested for homogeneous variance using Bartlett's tests The treatment groups were found to have homogeneous variances for all of the measured variables. For both experiments, the control group was compared to the other groups using Dunnett's test. All statistical computations were made according to the procedures described by Gill (1978) . Computations were made using Toxstat (Gulley et al., 1985) software. The Student’s t-test was used to determine if data from the two experiments could be analyzed together. It was important to combine the data from the two experiments so that the ganglioside profile at 4 days post-dosing could. be compared with the ganglioside profiles at 7, 14 and 21 days post-dosing. It was determined from the Student’s t-test that the untreated control groups from Experiments 1 and.2 could.be combined for all variables tested. RESULTS Clinical assessment of DFP-treated birds- As shown in Table 5, only one bird was showing signs indicative of OPIDN at 4 days post-DFP administration. By 7 days post-dosing, three of four birds were showing signs of mild ataxia and by 14 days post-dosing all birds were showing signs of moderate to severe ataxia. By 21 days post-DFP administration, the birds were exhibiting signs of paralysis. Table 5. Clinical assessment of adult chickens administered a single subcutaneous dose of DFP (1 mg/kg body weight). Days Post-DFP Number of Birds‘ Degree of Ataxia“ 4 1/4 3.0 7 3/4 2.0 .t 0.65“" 14 4/4 4.8 i 0.57 21 5/5 7.2 i 0.51 Number of birds exhibiting ataxia/number of birds in the treatment group. “ None (0); mild (1-2); moderate (3-4); severe (5-6): paralysis (7-8). ‘“Data expressed as mean i standard error of the mean. Experiment 1- The concentrations of protein, total. lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid in the hindbrains of control and DFP-treated chickens Tat 56 57 7, 14 and 21 days post-dosing are shown in Table 6. The treatment groups did not differ significantly from one another with respect to any of these variables. The relative percent distribution of gangliosides in the hindbrains of control and DFP-treated chickens at 7, 14 and 21 days post—dosing is shown in Table 7. The value of GM4 on day 7 was significantly lower than the control value. The value of GM4 increased after day 7 such that its values on days 14 and 21 were not significantly different from the control value. The decrease in the relative level of GM4 on day 7 is depicted in the representative chromatogram shown in Figure 8. The relative level of Gle also changed with time. The value of Gle on day 7 was similar to the control value. However, from day 7 to day 21 the value of Gle increased linearly such that its value on day 21 was significantly higher than the control value. The line fit to these points had a correlation coefficient of 0.99 and a slope of +0.47. Experiment 2- The wet weight concentrations of protein, total lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid in the hindbrains of control, DMSO-treated, parathion-treated and DFP-treated chickens at 4 days post-dosing are shown in Table 8. The treatment groups did not differ significantly from one another with respect to any 58 Table 6. Concentrations of protein, total lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid per gram wet weight (gww) in the hindbrains of control and DFP-treated chickens at 7, 14 and 21 days post-dosing. Control DFP-Treated Days Post-Dosing 7 14 21 Protein 69.1‘ 81.0 74.0 76.6 (mg/gww) 110.3 18.9 18.9 17.9 Total 288 305 282 270 Lipid 1 32 1 28 1 28 1 25 (mg/9W) Total 17.9 21.5 20.6 19.4 Chol. 11.5 11.3 11.3 11.1 (m9/gWW) Lipid 2430 2448 2808 2901 Phos. 1340 1294 1294 1263 (ug/gww) sialic 221 210 274 263 Acid 1 28 1 24 1 24 1 22 (HQ/9W) t Data expressed as mean 1 standard error of the mean. 59 Table 7. Percent distribution of ganglioside-bound sialic acid in the hindbrains of control and DFP—treated chickens at 7, 14 and 21 days post-dosing. Ganglioside Control DFP-Treated Days Post-Dosing 7 14 21 GM4 6.59“ 1.69“ 3.08 3.20 11.37 11.19 11.19 11.06 GM1 3.39 2.82 3.46 2.87 11.05 10.91 10.91 10.82 GD3 13.76 19.82 15.36 12.38 13.32 12.87 12.87 12.57 GDla 12.89 17.35 14.40 15.51 11.37 11.19 11.19 11.06 GTla 5.44 6.00 6.75 4.66 +GD2 10.79 (10.68 10.68 10.61 Gle 10.78 10.22 10.01 8.51 11.21 11.05 11.05 10.94 Gle 36.24 31.17 33.70 35.33 14.56 (13.95 13.95 13.53 Gle 10.72 10.93 13.25 17.54“ 12.05 11.78 11.78 11.59 a Data expressed as mean + standard error of the mean. “Significantly different from control value at p<0.05. 60 GM4 MAJ ...___> “' GM1 GD3 I” T“ 1* Inn GDla . " *‘ CID " GTla D +002 1 Gle 4» if. ...: a Gle - Gle a. = = 9- «— GQ’ :4: ___ ___ 2:: .__ > W O U (D Figure 8. Thin-layer chromatogram of ganglioside extracts from the hindbrains of (A) control and DFP-treated chickens at (B) 7, (C) 14 and (D) 21 days post-DFP administration. The bovine standard is represented by (S). The arrows indicate gangliosides which were found to differ significantly from controls. 61 Table 8. Concentrations of protein, total lipid, total cholesterol, lipid phosphorus and ganglioside-bound sialic acid per gram wet weight (gww) in the hindbrains of control, DMSO-treated (vehicle control), parathion-treated (negative control), and DFP-treated (test group) chickens at 4 days post-dosing. Control DMSO Parathion DFP Protein 88.8' 89.3 87.3 88.9 (mg/gww) 12.7 13.8 12.7 12.7 Total 288 300 306 269 Lipid 1 15 1 21 1 15 1 15 (mg/9W) Total 19.8 18.8 19.1 19.6 Chol. 10.7 11.0 10.7 10.7 (mg/9W) Lipid 2461 2388 2520 2225 Phos. 1 65 1 92 1 65 1 65 (“Q/QWW) Sialic 326 313 374 448 Acid 1 37 1 52 1 37 1 37 (Hg/9W) Data expressed as mean 1 standard error of the mean. 62 of these variables. The relative percent distribution of gangliosides in the hindbrains of control, DMSO-treated, parathion-treated and DFP-treated chickens at 4 days post-dosing is shown in Table 9. The ganglioside profiles for these four treatment groups were not significantly different. A representative chromatogram of these four treatment groups is shown in Figure 9. Because there was a linear increase in the value of Gle from days 7 to 21 post-DFP exposure, the changes in the values of the other gangliosides during this same period were examined. Two gangliosides, GD3 and Gle, were also found to undergo a linear change from day 7 to day 21. The line fit to the values of GD3 during this period had a correlation coefficient of 0.99 and a slope of -0.53. The line fit to the values of Gle during the same period had a correlation coefficient of 0.99 and a slope of +0.30. A plot of the relative proportions of the gangliosides in the hindbrains of control and DFP-treated chickens at 7, 14 and 21 days post- DFP administration is shown in Figure 10. Combined data from Experiments 1 and 2- According to the results of the Student's t-test, the untreated control groups from the two experiments were not significantly different. The data from the two experiments were therefore combined (Table 10) so that possible trends in 63 Table 9. Percent distribution of ganglioside-bound sialic acid in the hindbrains of control, parathion-treated (negative control), DMSO-treated (vehicle control) and DFP-treated (test group) chickens at 4 days post-dosing. Ganglioside Control DMSO Parathion DFP GM4 4.22' 3.47 2.66 5.11 11.01 11.43 11.01 11.01 GM1 5.72 4.42 4.90 5.76 10.70 10.99 10.70 10.70 GD3 19.42 19.59 18.00 20.96 11.57 12.22 11.57 11.57 GDla 17.17 18.01 18.33 18.74 11.11 11.01 11.11 11.11 GTla 5.76 5.85 5.74 6.15 +GD2 10.51 10.70 10.51 10.51 Gle 8.81 9.55 8.88 9.91 10.49 10.70 10.49 10.49 Gle 27.27 27.29 29.41 25.24 12.06 12.92 12.06 12.06 Gle 11.65 11.82 12.10 8.15 11.57 12.22 11.57 11.57 h Data expressed as mean + standard error of the mean. 64 GM4 I. 0' GM1 . . * II GD3 u G . . GDla - ' I GTla - - . . . +GD2 . “A * a-I 1 Gle .... ‘ __, - $ Gle ‘- Gle 2 2 I 2 . GQ’ Lfl __, __ ‘__ A B C D 8 Figure 9. Thin-layer chromatogram of ganglioside extracts from the hindbrains of (A) control, (B) parathion-treated, (C) DMSO-treated and (D) DFP—treated chickens at 4 days post-dosing. The bovine standard is represented by (S). 65 Figure 10. The relative percent distribution of GM4, GM1, GD3, GDla, GTla + GD2, Gle, Gle and Gle in the hindbrains of control chickens (day 0) and DFP-treated chickens at 7, 14 and 21 days post-DFP administration. 66 .oH ar=m_1 £69 1.... £5 1.? :59 ....T New + £5 1T flow ..T 80 ..... .29 IT v20 II. ZQEEWWEEQ $0-558 m>