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FINES will be charged if book is returned after the date stamped below. :an ; q 1992 um. 0'51???" gs 113‘ 42% ”$1998 ASSESSMENT OF THE EUROPEAN FERRET AS A MODEL SPECIES FOR ORGANOPHOSPHATE-INDUCED DELAYED NEUROTOXICITY by Anne M. Kogut A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1986 ABSTRACT THE ASSESSMENT OF THE EUROPEAN FERRET AS A MODEL SPECIES FOR ORGANOPHOSPHATE—INDUCED DELAYED NERUOTOXICITY by Anne M. Kogut Organophosphate-induced delayed neurotoxicity was studied in the European ferret (n;_ putorius furg) to show species sensitivity, assess clinical signs and histopathologic lesions, and to evaluate the use of the ferret as a Inodel species. In dermal and oral tests, adult male ferrets were given a single dose of tri—o—toyl phosphate (TOTP). In each test, 10 ferrets per group were treated with 0, 250, 500, or 1000 mg TOTP/kg body weight. At 48 hours post—treatment, half the animals in each group were sacrificed to assess whole brain neurotoxic esterase activity. The remaining ferrets were observed and received neurologic examinations for 58 days. All ferrets treated dermally with 1000 mg TOTP developed clinical signs of delayed neurotoxicity ranging from ataxia to partial paresis. Ferrets administered 250 and 500 mg TOTP developed variable degrees of ataxia. None of the animals dosed orally developed progressive delayed neurotoxic signs. Axon degeneration in the dorsal spinocerebellar tracts was the most consistent lesion. Neurotoxic esterase activity was maximally inhibited at 46% of control activity 48 hours post-exposure. Results suggest that. the ferret can be used as a model for organophosphate—induced delayed neurotoxicity. ACKNOWLEDGEMENTS I wish to thank the members of my family, my mom, and my sister Monica, and my fiance Jim. Thanks for all your support, love, and understanding. Jim's constant encouragement, love, and emotional support (not to mention all his help with the ferrets) kept my hopes and goals in sight. I would also like to thank Dr. Richard Aulerich, my major professor, whose constant guidance and insight allowed me to keep my chin up and finally finish my research. I extend this gratitude to the other two members of my committee, Dr. Steven Bursian and Dr. Duke Tanaka. Dr. Bursians knowledge and interest in my area of research helped me solve some of the roadblocks I[ encountered along the way. Dr. Tanaka not only encouraged. me to develop my talents as a student and individual, but also taught me invaluable histopathological techniques and interpretations. Many others have made this accomplishment possible. I would not have been able to conduct all my laboratory work without the assistance of Kay Trosko. Thanks also goes to Angelo Napolitano and the rest of the people at the Experimental Fur Farm for their assistance in animal care, and to Ms. Carol Daniel for typing this manuscript. Finally, I would like to extend my deepest gratitude to the Office of Radiation, Chemical, and Biological Safety, ii not only for financial assistance, but mostly for the friendships of everyone associated with the ORCBS. iii TABLE OF CONTENTS Page Acknowledgements ..................................... ii List of Tables ....................................... vi List of Figures ...................................... v Introduction ......................................... 1 Review of Literature ................................. 2 The Ferret ......................................... 2 Natural History and Care ......................... 2 Use in Research .................................. 3 Tri-ortho-Toyl Phosphate ........................... 5 Organophosphate-Induced Delayed Neurotoxicity ...... 7 Historic Background .............................. 7 Species Susceptibility and Variability ........... 9 Mechanism of Action .............................. 12 Clinical and Pathological Manifestations ......... 13 Materials and Methods ................................ 16 Animal Selection and Care .......................... 16 Test Procedures .................................... 17 Single Dermal Dose ............................... 19 Single Oral Dose ................................. 20 Weights and Measurements ........................... 21 Clinical Evaluations and Tests ..................... 21 General Observations of Gait, Posture, and Habits ............................ 21 Spinal Cord Reflexes ............................. 22 Postural Reaction ................................ 24 Histopathological Techniques ....................... 25 Thionin Stain ................................... 26 Modified Fink-Heimer Technique .................. 26 Speilmeyer Method ............................... 27 Statistical Analysis ............................... 28 Results .............................................. 28 Body Weight Changes and Feed Consumption ........... 29 Neurotoxic Esterase (NTE) Activity ................. 34 Clinical Observations .............................. 34 Histopathology ..................................... 46 Discussion ........................................... 55 iv Conclusions .......................................... 65 References ........................................... 66 Table Table Table Table Table Table Table LIST OF TABLES Susceptible and non-susceptible species to organophosphate-induced delayed neurotoxicity .................. Ferret diet ............................ Description of rating scale and criteria used in assessing organophosphate-induced delayed neurotoxicity .......................... Effect of dermal and oral administration of TOTP on neurotoxic esterase activity in the ferret .......................... Description of clinical response ratings ....................... Clinical assessment of ferrets administered tri-ortho-tolyl phosphate .............................. Neurological evaluations of ferrets administered tri-orthotolyl phosphate ............... vi ll 18 23 35 36 42 44 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. ll. 12. LIST OF FIGURES Reaction of TOTP to form the toxic metabolite saligenin cyclic phosphate .................... Effect of dermal administration of TOTP on feed consumption ......... Effect of oral administration of TOTP on feed consumption ......... Effect of dermal administration of TOTP on body weight .............. Effect of oral administration of TOTP on body weight .............. Control ferret displaying characteristic posture .............. Arched back of ferret administered 1000 mg TOTP ........... Sliding gait and sprawled legs of a TOTP—treated ferret .............................. Hindlimbs of control and TOTP-treated ferrets ................ Cross section of lumbar spinal cord of a ferret administered 1000 mg TOTP ........................ Dorsal spinocerebellar tracts of a ferret administered 1000 mg TOTP ............................. Longitudinal section of dorsal spinocerebellar tracts in the lumbar region of a spinal cord in a control ferret ................. vii Page 30 31 32 33 37 38 39 41 47 47 49 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure l3. 14. 15. 16. 17. l8. 19. 20. 21. 22. Page Longitudinal section of dorsal spinocerebellar tracts in the lumbar region of the spinal cord in a control ferret .................. 49 Longitudinal section of dorsal spinocerebellar tracts in the lumbar spinal cord of ferret number F—2 administered 1000 mg TOTP ...... 50 Longitudinal section of dorsal spinocerebellar tracts in the lumbar spinal cord of ferret number F-2 administered 1000 mg TOTP ...... 50 Longitudinal section of dorsal spinocerebellar tracts of ferret number F-13 administered 1000 mg TOTP ................................... 51 Longetudinal section of dorsal spinocerebellar tracts of ferret number F—13 administered 1000 mg TOTP ..... 51 Cross section of lumbar spinal cord of a control ferret .................. 52 Cross section of lumbar spinal cord of a ferret administered 1000 mg TOTP/kg ........................... 52 Cross section of cervical spinal cord of a control ferret ........... ......................... 53 Neuron from ventral horn of cervical spinal cord from a control ferret ............................ 53 Neuron in the ventral horn cervical spinal cord of a ferret treated with 1000 mg TOTP/kg ................................ 54 Neuron in cervical spinal cord of a ferret treated with 1000 mg TOTP/kg ...................... 54 viii INTRODUCTION The increased concern about the effects of chemicals and. environmental contaminants (n1 human. health Imus prompted many toxicological evaluations. Organophosphates are some of the most widely used compounds in agriculture and industry and have been related to potential health problems. Organophosphate—induced delayed neurotoxicity (OPIDN) is just one possible consequence of exposure to organophosphates. This delayed neurotoxicity is defined as nerve damage with delayed onset or toxic distal axonopathy (Shaumberg and Spencer, 1979). The clinical effect is characterized by an initial delay period of 14 to 21 days, after which development of ataxia that progresses to paralysis is observed. Morphological changes in animals that exhibit this condition include axonal degeneration with subsequent Wallerian degeneration of the central and peripheral nervous system. Compounds such as 'tri—ortho—toyl phosphate (TOTP), phenylphosphonothionate (leptophos), mipafox, trichlofon, and O—ethyl 0-4 nitrophenyl phenylphosphonothioate (EPN) have been implicated in the production of OPIDN in animals and humans. Factors that influence the neurotoxic effect involve the form anui purity of time test compound, dosage, frequency and duration of exposure, route of administration, metabolism, and toxicokinetics (Abou—Donia, 1981). Species sensitivity is another factor that influences the occurrence of organo— phosphate-induced delayed neurotoxicity. Common laboratory animals such as the mouse and rat have been found to be relatively insensitive to OPIDN. The chicken is presently the species of choice for OPIDN testing, but the validity of extrapolation of data to man has been questioned. The cat has also been utilized for OPIDN testing, but the increased disfavor towards the use of pet species in research has led to the search for more suitable species for toxicological evaluations. The necessity for another species sensitive to OPIDN prompted this study on the European ferret (Mustela putorius figgg). The objectives of this study were: 1. To assess the sensitivity of the ferret to OPIDN by clinical observations. 2. To determine histopathological changes related to OPIDN. 3. To assess the use of the ferret as a model species for OPIDN. LITERATURE REVIEW The Ferret Natural History and Care The European ferret belongs to the order Carnivora and the family Mustelidae. Two color strains are found, one of which is the fitch color, characterized by a brown and yellow patterned coat. The second, the ruby-eyed albino, is recessive to the fitch variety. The ferret was introduced into the Americas in the 19th century where it came from its native lands of Northern Africa, Europe, and Asia (Willis and Barrow, 1971). Ferrets have been used in the past for rabbit hunting but have become a popular choice in recent years for laboratory animals and pets. However, some states have laws that prohibit possessing ferrets because of a concern about proliferation of this species in the wild and possible damage to poultry farms (Williams, 1976). Male ferrets weigh between 1350 and 2100 gms, but show a marked seasonal weight change; there is a loss in weight during the breeding season and a gain during the fall (Williams, 1976). Their body length is approximately 30 cm and they have a lifespan in commercial breeding operations of 2-5 years. Since the ferret is a carnivore, it requires a high amount of protein in its diet. A large variety of foods such as commercial and conventional mink diets or commercial dog and cat food provide suitable nourishment. The ferret has a seasonal breeding period that extends from March to August. The female exhibits delayed ovulation (Hahn and Wester, 1969). The gestation period is 42 days and two litters of 3 to 13 kits per year are normal (Asdell, 1946; Farris, 1950). Use in Research The ferret has become a popular choice for a research animal and has been used in such research areas as virology, with an emphasis on canine distemper (Green, 1945), influenza (Perrin and Oliphant, 1940), gastroenterology (Pfeiffer and Weibel, 1973), and endocrinology (Rieger and Murphy, 1977). The occurrence of spontaneous genital malformations has made the ferret a desirable species for teratological research (Haddad _ei a_1., 1975; Elizan _et a_1., 1964). In a recent investigation. of the ferret's haematology, serum. chemistry, pathology, and reproduction has concluded that the ferret is a suitable species for toxicological. evaluations (Hf new and experimental drugs (Thornton e_t _a_l_., 1979). The ferret has also become a potential animal model for cardiological research on anatomical relationships of the aortic arch vessels (Willis and Barrow, 1971). The increasing use (n5 non-rodent species anui consequent demands and controversies over the use of primates and dogs in research. has led. to the jpossible use (Hf the ferret. as a non-rodent species. The ferret is also desirable as a laboratory animal because it is very adaptable to human handling, can be housed in standard laboratory cages, requires no exercise space, and can be raised in colonies (Hahn and Wester, 1969). Ferrets are also excellent lab animals because of their relatively short gestation period and large litter size and because their breeding season can be regulated through manipulation of the photoperiod (Willis and Barrow, 1971). Tri—Ortho-Toyl Phosphate (TOTP) TOTP is a tri-aromatic organophosphorus ester which is used as a pflasticizer in lacquers and varnishes, and also as a gasoline additive, pesticide and flame retardant (Fassett, 1967). Other synonyms for this compound are: tri-ortho- cresyl phosphate (TOCP), tri-2—toyl phosphate, o-cresyl phosphate, and tri—2—methylphenyl phosphate (Sax, 1984). TOTP is 21 colorless liquid Rudd) a molecular weight of 368.39 and melting and boiling points of -30°C and 410°C, respectively. 11: is insoluble 1J1 water Ibut i1; soluble in alcohol and ether. Cavanagh (1973) noted that. the presence of .2 cut more 2-methy1 substituted benzene rings was necessary for toxicity and that the liver played an important role in activating the parent compound. TOTP is 51 relatively' inactive: parent molecule that does not induce acute cholinergic or neurotoxic effects, but requires bioactivation 1x) produce neurotoxicity (Casida gt_gl., 1961). This conversion is a two step reaction which involves a mixed-function oxidase hydroxylation of a methyl group and then a cyclization reaction, catalyzed by albumin (Figure l). TOTP and its metabolite, saligenin cyclic phosphate, are used as model chemicals against which other compounds are tested for delayed neurotoxicity potential (Baron, 1981). Saligenin cyclic phosphate was found to be five times more neurotoxic than TOTP after oral administration to chickens (Bleiberg and Johnson, 1965). FIGURE 1. Reaction of TOTP to form the toxic metabolite saligenin cyclic phosphate. madmmmomm UHQUWU ZHZMUHflflm ZHZDQAG NmflDHNO ZCHBUZDQ DMNHZ A ABOB Organophosphate-Induced Delayed Neurotoxicity Historic Background OPIDN has been recognized in both humans and animals for over 50 years. The extensive use of organophosphates in industry and agriculture has increased the chances of toxicity to man, however, most of the human outbreaks of delayed neurotoxicity have been accidental. OPIDN was first recognized by Lorot in 1899 following the treatment of tubercular patients with phosphocresote (Metcalf, 1982). During the late 1920's and 1930's, an outbreak of polyneuritis existed throughout the midwest and southwest United States. The causal agent was found to be TOTP (Smith gt; 31., 1930). An 80% alcoholic extract of Jamaican ginger was adulterated with 2.5% TOTP and was sold over the counter during the U.S. Prohibition Era. The condition that developed from the ingestion of the alcohol extract became known as "ginger jake syndrome" and. was characterized iby 51 delay' period of 10—21 days, after which symptoms of weakness in fingers, foot drop, and partial paralysis developed (Baron, 1981). Over 20,000 people experienced various degrees of paralysis resulting from an average exposure to 30-40 mg TOTP/kg body weight. Various other incidents of poisoning from ingestion of food or drink adulterated with cresyl phosphates have been described. In 1936, 60 people were left permanently paralyzed in Napal after lubricating oil containing 0.4% TOTP was accidently added to soybean oil (Metcalf, 1982). Also in the 1930's, a widely sold abortifacient of parsley extract (Apiol) produced OPIDN in 90 women in Holland, Germany, France, Yugoslavia, and Sweden (Davies, 1963). This extract contained 20-50% TOTP. Between 1939 and 1945, many German and Swiss soldiers became affected when lubricating oil or contaminated cooking oil was used for cooking (Metcalf, 1982). Another large epidemic of delayed neurotoxicity poisoning occurred 1J1 Morocco III 1959 where over 10,000 people became affected after using cooking oils adulterated by aircraft engine oils containing approximately 3.0% cresyl phosphate (Smith and Spaulding, 1959). Dogs also reportedly showed neurotoxic symptoms in this incident. The outbreaks of OPIDN were not confined only to TOTP, in that other chemicals were found to induce neurotoxic symptoms. In 1953 workers manufacturing a systemic insecticide called mipafox developed acute organophosphate poisoning symptoms of vomiting, nausea, cramps, and general. muscular weakness (Bidstrup gt 31., 1953). These acute symptoms subsided after 4 days, but were subsequently followed by leg weakness and flaccid paralysis characteristic of OPIDN. During the 1950's and 1960's, thousands of chemicals had been evaluated for use as pesticides and some were found to cause delayed neurotoxicity in animals and man. One of these compounds, leptophos, continued to be marketed even after tests confirmed the production of OPIDN in hens (Metcalf, 1982). After large applications of substantial quantities of leptophos in Egypt, over 1300 water buffalo died from OPIDN and widespread human poisoning developed (Abou-Donia 22.21:! 1974). The problem with leptophos prompted the U.S. Environmental Protection Agency to revoke all tolerance levels for residues of this pesticide in foods (Casida and Baron, 1976). Testing requirements for delayed neurotoxicity have been in effect in the U.S. since August 1978 (Federal Register, 1978). This testing applies to all pesticides registered for use in the United States if the active ingredients, metabolites, degradation products, or impurities cause acetylcholinesterase depression or are structurally related to substances that are known to induce delayed neurotoxicity. The World Health Organization (1976) has classified the hazards of delayed neurotoxicity as a dose response relationship that involve exposure of individuals to occupationally or accidentally high doses for short periods of time or exposure to low levels for an extended period of time. Investigations are now in progress to estimate "no effect levels" of exposure to these compounds. Species Susceptibility and Variability The production of OPIDN in animal studies designed to simulate human exposure is marked. species differences in susceptibility. The White Leghorn hen (Gallus gallus domesticus) tun; been used as ea model species because of its adaptability and availability. Abou-Donia (1981) has concluded that the attributes of the use of the chicken as a model for OPIDN include a complete correlation. with human response, in that the delay period, pathology and clinical signs are similar to humans, both sexes are susceptible, clinical signs are standardized and easy to grade, and there is an availability of extensive structure-activity data. CHM; domestic (EN: (Felis domestica) has also been suggested as a model species but three differences in response when compared to the chicken have been noted. These differences include a difficulty in protecting against acute poisoning, slower progression of delayed neurotoxicity, and the propensity for improvement in some animals (Abou—Donia §t_gl,, 1983b). The list of susceptible and non-susceptible species, shown in Table l, was compiled by Johnson (1975) and Baron (1981). The slow loris (Nycticebus coucany coucany) was found to be affected by repeated dermal applications of tricresyl phosphate (TCP) (Ahmed and Glees, 1971). Triaryl phosphate poisoning was exhibited in cattle exposed both naturally and experimentally (Beck _gt .gl., 1977). Rats, guinea pigs, and Japanese quail are all insensitive to known delayed neurotoxins when administered in single doses (Johnson, 10 Table 1. Susceptible and non-susceptible species to organo- phosphate-induced delayed neurotoxicity (Johnson, 1975; 1981). Susceptible Non—susceptible Man Rat Baboon Mouse Squirrel Monkey Rabbit Water Buffalo Guinea Pig Horse Hamster Cow Gerbil Sheep Japanese Quail Pig Dog Cat Slow Loris Chicken Duck Pheasant Turkey Partridge ll 1975). The differences in susceptibility may be due to inadequate absorption of the neurotoxin, enhanced metabolism and elimination, and/or the unique structure of the nervous system of individual species (Bursian §E_gl,, 1983). Immature animals of a sensitive species have been found to be less susceptible than adults to OPIDN (Cavanagh, 1973). Mechanism of Action The initial studies on delayed neurotoxicity showed that the site of action was a defined area in the nervous system. Modell e_t 11. (1946) and Koelle and Gilman (1946) hypothesized that the paralytic effects seen after administration of di-isopropyl fluorophosphate (DFP) were due 11) prolonged inhibition (n3 acetylcholinesterase. Another esterase besides acetylcholinesterase was implicated by the studies of Baron and Cassida (1962). Subsequently, phosphorylation sites present. in. the ibrain enKi spinal cord were identified (JOhnson, 1969). This "neurotoxic esterase" (NTE) was foumd to be inhibited by known delayed neurotoxins i_n Egg and was convincingly determined to be the site for initial neurotoxic activity when certain protective compounds were bound to the esterase and prevented delayed neurotoxic effects (Johnson, 1970). Thus, it vnus determined. that 'the nature of the group binding to the active site and the binding site itself resulted in the neurotoxic response. Johnson (1974) further demonstrated that the binding 1x3 NTE by El delayed neurotoxic compound induced £1 hydrolysis 12 of an ester or amide bond, resulting in an ionized acidic group. This time dependent process was termed "aging" and was found not to occur with non—delayed neurotoxic compounds. It was hypothesized that the charged group could alter membranes, thus inducing initial biochemical lesions. A recent interpretation of the mechanism of action suggests that initiation of OPIDN can be monitored by the inhibition of catalytic ability (Johnson, 1982). Two events can occur after this initiation. Either the inhibited NTE "ages" and the modified protein has a deleterious degenerating effect on the long axons, or the binding of NTE does not occur, resulting in no aging effect and a normal response. The processes following initiation which lead to development of neuropathy have not been established. Assays have been used to predict the delayed neurotoxic potential by the amount of NTE inhibition. In hens, it was found that for single dose tests, 75% inhibition initiated neuropathy and 45-65% inhibition produced neuropathy in chronic tests (Johnson, 1982). Clinical and Pathological Manifestations The acute toxic clinical signs of OPIDN poisoning are characteristic of cholinergic poisoning and include tremors, lacrimation, salivation, diarrhea, and other effects of parasympathetic stimulation, OPIDN 1i; characterized kn? three significant features unrelated to the acute signs (Baron, 13 1981). First, a delay occurs in the interval following acute poisoning. During this time, no evidence or signs of poisoning exists, but, following this period, a progressive ataxia occurs. Second, species differ in their susceptibility ‘to delayed neurotoxins. Finally, a large variety of organophospahtes and other chemicals may' cause: the same Ior similar lesions. Clinical signs are similar in all susceptible species. Not only are the effects dose dependent, but route of administration also plays a najor role in delayed neurotoxicity (Baron, 1981). Clinical signs are not seen until 8 to 10 days or up to 21 days after exposure (Stuart and Oehme, 1982). Weakness and ataxia develop in the lower limbs and may progress to paralysis. In severe cases, the upper limbs may also be affected (Johnson, 1980; Gordon, 1983). The weakness can then be followed by what is described as "toe drop" in man, which produces a characteristic high stepping gait (Smith. gt__gl., 1930). It progressive flaccid paresis of the legs (hindlimbs in quadrapeds) with possible effects on the forelimbs has been observed (Stuart and Oehme, 1982). The onset of paralysis in man is accompanied by abnormal sensory experiences such as a burning and tingling sensation. in the feet (Cavanagh, 1973). The Ihindlimbs are always more affected than the forelimbs in all species. Certain species have been noted to express characteristic responses in relation to OPIDN. For example, cats were noted 14 to have an abnormal response to the stroking reflex and a reluctance to jump (Taylor, 1967). Recovery' may occur in less severe cases and proceeds in the reverse order from which the motor deficits occurred (Stuart and Oehme, 1982). In man, recovery is slow and often, in severe cases, seldom complete, with residual ataxia present (Metcalf, 1982; Takade, 1982). The morphological lesion most associated with OPIDN is best described as a "dying-back" process or Wallerian degeneration of the long axons of the peripheral nerves and both ascending and descending tracts of the spinal cord (Bradley, 1976; Miller, 1983). Demyelination. was initially thought to 1x2 the primary lesion (Lillie and Smith, 1932) but it was later demonstrated that myelin degeneration was secondary to axon degeneration (Cavanagh, 1954). The degenerative lesions are most frequently observed in the larger diameter axons and can. be detected histologically at the same time as clinical signs are observed (Baron, 1981). Further, axonal changes appear to be well advanced before the myelin begins to disrupt (Baron, 1981). Abou-Donia and Graham (1979a) noted in chickens that lesions in the pmmipheral nerves occurred earlier than those in the spinal cord and that evidence of regeneration in the peripheral nerves was present. Also, the most consistent histological changes observed were degeneration of the anterior columns 15 of the thoracic and lumbar spinal cord. In the cat, histological changes consisted cfif swelling, vacuolation, and fragmentation of axons and myelin sheath (Abou-Donia _e_t_ a_1., 1983b). These lesions were located in the gracile and spinocerebellar ascending tracts (ME the posterior columns of the cervical spinal cord and also in the lateral corticospinal descending' tracts of time thoracic anui lumbar regions. Although evidence has been presented suggesting that no nerve cell damage occurs (Cavanagh and Keller, 1979), other evidence indicates nerve cell damage. Large numbers of chromalytic neurons in the lumbar regions of the spinal cord taken from a slow loris treated with TCP have been observed (Ahmed and Glees, 1971). These nerve cells are characterized by a pale cytoplasm, eccentric position of the nucleus, and the peripheral arrangement of the nissl substance. MATERIALS AND METHODS Animal Selection and Care Eighty adult male, agouti-colored, European ferrets (Mustela putorius furo) were obtained from Marshall Research Animals, North Rose, New York. The animals were immunized against canine distemper anmi housed indoors at time Michigan State University Experimental Fur Farm. Adult (1-3 years old) male ferrets were used because of the greater sensitivity of adult animals to delayed neurotoxicity (Barnes and Denz, l6 1953) and to eliminate any sex differences in response to the neurotoxin. In the test room, the ferrets were housed two per cage in galvanized wire cages (61 (L) x 30 (W) x 46 (H) cm) with attached feed and water cups. The cages were suspended 69 cm above the floor. The ferrets were subjected to ambient temperature and a 12 hour light/dark schedule. Ventilation was provided by wall fans and ceiling vents. Wood shavings, changed periodically, were spread under the cages to facilitate animal waste disposal. The ferrets were fed the diet shown in Table 2, which in previous studies (Bleavins and Aulerich, 1981) has been demonstrated to sustain ferrets in good health. Food and water were provided gg_libitum. Test Procedures Pre-test procedures for both the dermal and oral trials included a two week acclimation period during which the animals were weighed weekly and observed daily. Only healthy ferrets were used in the tests. A total of 40 animals were used for each trial. Since the animals were housed two per cage, cages were randomly assigned to a treatment group. Both animals in each cage were given the same treatment but the animals were assigned a unique number which was identified by the ferrets weight and distinctive color markings. There were four treatment groups consisting of 10 animals per group. The treatments 17 UT“ ”‘4 m" '7’” Table 2. Ferret diet. Ingredients Percentage Commercial Mink Cereal 20% Duck By-Producta 24% Ocean Fish Scrap 15% Beef Tripe 8% Beef Trimmings 4% Beef Liver 4% Beef Lungs 4% Added Water 21% a For the oral test, 24% chicken by-product was substituted for the duck by—product. 18 in. both ‘tests consisted of El control (vehicle only), 250, 500, and 1000 mg TOTP/kg body weight. Animals in groups were cared for in the same way. The day jprior' to» dosing, the animals were 'weighed to obtain an approximate weight in order to determine the quantity of TOTP required for dosing. Approximately 20 minutes before dosing and daily for 10 days thereafter all animals were given 1.5 mg of atropine sulfate (Professional Veterinary Laboratories, Minneapolis, MN) tut subcutaneous injection in the back of the neck to prevent possible illness or death due to the initial cholinergic effects of TOTP (Baron, 1981). Single Dermal Dose The amount of TOTP (Eastman Kodak Company, Rochester, New York) required to yield the desired dose levels of 250, 500, and 1000 mg TOTP/kg body weight was calculated as follows. Using a density factor of 0.862 for TOTP, 5.4, 10.8, and 21.6 ml of ‘TOTP ‘were dissolved in 19.6, 14.2, and 3.4 IE1 of 95% ethyl alcohol to yield the desired concentrations of 250, 500, and 1000 mg TOTP/ml, respectively. One ml/kg body weight of TOTP was applied to a shaved area (approximately 10 cm2) on the back of the neck of each ferret. Ethyl alcohol was applied. to the controls. The solution. was allowed to be absorbed through the skin before the animals were returned to their cages to ensure that the compound was not consumed or rubbed from the skin. After dosing, the animals were 19 observed closely for 24 hours to note any abnormal behavior or adverse health effects. Five animals (one per cage) per treatment group were sacrificed by cervical dislocation 48 hours post-treatment for determination of whole brain neurotoxic esterase (NTE) activity. Brains were removed, weighed, and immediately frozen in dry ice, and subsequently analyzed for NTE activity, as described by Johnson (1977). The remaining 20 animals were observed daily for development of clinical signs typical of OPIDN and were given neurological examinations during the next 58 days. Single Oral Dose TOTP was mixed as described for the dermal trial, except corn oil replaced ethyl alcohol as the vehicle. The animals were dosed by gavage by placing a 3 (W) x 10 (L), 0.3 thick cm piece of clear plastic, with a 4 mm hole in the center, behind the canine teeth to hold the animals mouth open (Hornshaw, 1984). A 1.4 (ID) x 1.57 (OD) mm piece of polyethylene tubing, attached to an 18 gauge needle on a 3.0 m1 syringe was inserted through the hole in the plastic, down the esophagus, and into the animal's stomach. This procedure prevented the ferret from biting the tubing and also assured that the animal was administered the entire dose. As in the dermal trial, 20 ferrets (one from. each cage) were killed 48 hours post-dosing and the brains removed 20 for analysis of NTE activity. The remaining ferrets were observed daily for development of clinical signs of OPIDN and were given neurologic examination during the next 58 days. Weights and Measurements All animals were ‘weighed on 51 digital. balance (Fisher Scientific Co., Model #EWO—4010) when first obtained and at weekly intervals throughout the trial. To account for variations in daily feed consumption, feed intake was measured for 2 consecutive days each week during the test period. During the days feed consumption was measured, feed was placed in containers (12 oz. tuna cans) secured to the bottom of the cages, rather than provided on the feed grids, to avoid. wastage. An. amount. of feed, in excess of what a ferret would normally consume in a day, was placed in each container. These filled containers were weighed and then placed in the cages. At approximately the same time the next day, the containers were removed, weighed, amount of feed consumed recorded, and any uneaten feed discarded. Fresh feed was placed into the containers and the procedure repeated. In the event of spilled or wasted feed, the data were discarded. Clinical Evaluations and Tests General observation of gait, posture and habits Beginning approximately 51 week after administration of TOTP, the animals were evaluated three times a week for 21 development of clinical signs typical of delayed neurotoxicity. Each animal was allowed to walk freely on a flat level wooden surface for 3-5 minutes. During this "walking time" the animals were observed for abnormalities in posture, gait, and general behavior. Normal ferrets ‘walked. in 51 crouched position with their heads pointed towards the ground, sniffing. Their gait. was SlOW’ or fast, but very coordinated. Those animals showing any abnormalities during the observation period were subjected to further neurological tests similar to those used to evaluate the cat for delayed neurotoxicity (Abou—Donia g£_gl., 1983b). Each animal was subjectively rated on a scale from 0-6 for general observations. Those animals that received a score of Eq_ the control ferret, the treated animal shown in Figure 7 has an arched back, characteristic of the .R3 response. Figure £3 shows 51 ferret with 51 sprawled, sliding 34 Table 4. Effect of dermal and oral administration of TOTP on neurotoxic esterase activity in the ferret. Neurotoxic esterase activity nmole product/min/gram tissue) Dose (mg/kg) Dermal Oral 0 1351a i 53b 1257 i 112 250 1142 i 39 966 f 176 (18%)C (23%) 500 1019 i 63 797 i 148 (25%) (37%) 1000 735 f 77 931 i 46 (46%) (26%) a Mean i standard error. Based on 5 animals per treatment group. Numbers in parentheses denote percent inhibition of NTE activity when compared to the appropriate control. 35 Table 5. Description of clinical response ratings. Response rating Observed clinical signs R1 R6 Muscle stiffness Slowed movements and careful placement of hindfeet Hopping movements instead of walking Some reluctance to walk Uncoordinated movements Stiff wobble walk High stepping walk Back arched during walking, with uncoordinated movements (Figure 3) Mild ataxia Unsteadiness in walk Sliding walk with sprawled legs Sits on haunches and falls over Occasional falling over during walking Moderate ataxia Sprawling walk (Figure 4) Falls over during walking Occasional dragging of hindfeet Difficulty in righting itself Severe ataxia Complete paralysis 36 FIGURE 6. Control ferret displaying characteristic posture. 37 FIGURE 7. Arched back of ferret administered 1000 mg TOTP. 38 FIGURE 8. Sliding gait and sprawled legs of a TOTP-treated ferret. 39 gait, characteristic of the R4 response. The differences in hindlimb support between a control ferret and a 1000 mg TOTP dermally—treated ferret is illustrated in Figure 9. The control animal on the right shows normal, strong hindlimb support, while the treated animal on the left exhibits minimal hindlimb support. The individual animal rating" responses for txfifli trials are summarized in Table 6. There was an ll-day delay period before the initial onset of any cflinical signs. Two ferrets (F2 and F13) administered dermal doses of 1000 mg TOTP showed the earliest clinical signs and progressed the quickest to advanced stages of neurologic deficits. The days showing the onset of responses indicate a slow progression of increased neurological deficiencies over time. None of the animals in the dermal trial, showing progressive responses, exhibited any signs of recovery from these deficits. Only three animals on the oral trial showed delayed neurotoxic signs (Table 6). However, these signs did not progress beyond an R2 response and diminished after 55 days. Results obtained from the additional neurologic examinations are shown in Table 7. The treated animals did not display abnormal locomotor or reflex signs until at least 10 days after general clinical signs ‘were: observed. Also, the animals did not show an abnormal response to the neurologic examination until the general observation response was at 40 FIGURE 9. Hindlimbs of control and TOTP-treated ferrets. 1D.aoo .e magmas Am H Nev Av H mmv Am H HNV Am H omv Am H may mm mm 0H ma HH ma In mm It mm Hm mm mm ma I: ma HH m I: mm mm mm mH mm me mm mm I: am mm OOOH AH H mmv II II II we mm «H I: it I: In mm mm II II I: ma mm 0H II II In me mm 0H I: In I: I: II mm oom QAH H va II In In I: Hm ma 2 it In I: II mm cm 4 I: II In I: ll em II II In I: II mm It In II II In em omm II II II I: II m I: II II II II o 1: II II II In mm I: II II II In ow II II II II In a o HmEHoo mm em mm mm Hm Hogans AHQmHoz woos AHcoEHOOHHIHmom.mmmUV mmcHHmH omcommom Houuom mx\maoa mEv omoo .mpmnmwosm HSHOHIOSHHOIHHH OOHOHmHaHEom muouuom mo HcmEmmmmmm HOOHGHHU .m OHQOB .m>m© mm Houwm emochHEHU noncommom o .msoum HQOEHOOHH Hog mHmEHcm m .Hcofiumouplumom mwmp .HOHHO eumocmum H CODE 9 .mmcHHmH oncommwu mo COHHQHHomop How m canoe mom m 32 3. H «S II II II om mm um I: II II II mm mH In I: II om Hm mm II II II II 1: ca II II II :1 II om coca .momcommon HEEHOQQD oozosm mHmEHcm oz oom .momcommom HmEHocom pozonm mHmEHcm oz omm .momcommon HOEHOCQO UOBOSm mHmEHQO oz 0 HOMO mm am mm mm Hm HOQEDQ HanOB woon AHSOEHOOHHIHmom mwmpv mmcHHcH oncommom Houuom mx\mBOB mEv omom AH.GOO .@ OHQMBV 43 Table 7. Neurological evaluations of ferrets administered tri-ortho-toyl phosphatearb. Dose (mg TOTP/kg Ferret Response ratings (days post-treatment body weight) number R1 R2 R3 R4 R5 Dermal Flexor reflex 1000 2 22 24 —- 44 —- 13 22 24 44 -- -- 35 24 42 —- -- -- 22 49 53 57 -- -- 38 42 47 51 -- -- Extensor-postural thrust and hindlimb walking 2 22 24 26 38 55 13 22 24 44 49 -- 35 34 42 53 -- -- 22 42 51 53 -- -- 38 38 40 51 -- -- Extensor-thrust reflex 2 22 24 26 38 55 13 22 24 44 49 -- 35 34 42 53 -- -- 22 42 51 53 -- -- 38 38 40 51 -- -- Hanging and grasping reflex 2 -— 28 30 34 -- 13 22 28 32 57 -- 35 42 53 -- -- -- 22 49 -- 53 -- -- 38 38 47 57 -- -- (Table 7, con't) 44 (Table 7, con't) Dose (mg TOTP/kg Ferret Response ratings (days post-treatment body weight) number R1 R2 R3 R4 Oral Flexor reflex 1000 37 36 —- -- -— 23 38 -- -- -— Extensor-postural thrust and hindlimb walking 37 38 —— —- —— 23 43 -- -— -— Extensor-thrust reflex 37 38 -- -- -- 23 43 -- -- -- Hanging and grasping reflex 37 33 —— -— -- 23 38 -- -- -- a Animals exposed to TOTP at doses below 1000 mg/kg body weight gave normal responses in the neurological tests performed and thus are not included in the table. b See Table 3 for description of response ratings. 45 least an R3. Animals that had abnormal flexor reflex responses showed a decrease in response time after the stimuli was administered. Abnormal extensor postural thrust and hindlimb walking response was characterized by the animal's tendency to set on its haunches (instead of standing on the hindlimbs), falling over, and showing a reluctance to walk. Extensor-thrust responses were the same as postural-thurst responses (Table 7). These animals exhibited. little tx> no resistance to exerted pressure on the footpads of the hindlimb. Abnormal responses to the hanging/grasping test were generally characterized by the lack of extraction of the claws and a limp posture of the hindlimbs. The animals in all treatment groups for both trials had normal responses to the wheelbarrow test. No abnormal responses to the proprioreceptive positioning and righting reactions were observed in the ferrets on the oral trial. Histopathology {Hue most distinctive differences noticed 111 the tissues were obtained by the Fink-Heimer 'method. Degeneration. was found in both animals administered dermal doses of 1000 mg TOTP and was located in the dorsal spinocerebellar tracts of the lumbar spinal cord (Figure 10). Degeneration of the long axons in these tracts was characterized by the presence of black, silver' impregnated, fragmented. axons (Figure .11). For comparison axons of the dorsal spinocerebellar tracts 46 FIGURE 10. Cross section of lumbar spinal cord of a ferret administered 1000 mg TOTP (84X, Fink-Heimer). FIGURE 11. Dorsal spinocerebellar tracts of a ferret administered 1000 mg TOTP (350x, Fink-Heimer). 47 from controls are shown in Figures 12 and 13. The axon fibers are intact and lack the fragmented appearance of degenerating fibers. Longitudinal sections of this same area in a 1000 mg TOTP/kg dermally-treated animal (F-2) are shown in Figures 14 and 15. The axon degeneration is denoted by the silver impregnation of fragmented nerve fibers. Animal F-13, also treated dermally with 1000 mg TOTP/kg, exhibited more extensive degeneration in these tracts (Figures 16 and 17). Lesions were not detected at the cervical levels of the spinal cord nor in animals administered 500 and 250 mg TOTP/kg. Observations of the tissue stained by the Speilmeyer method, which stains myelin, did not yield any discernible differences between treated and control animals. Myelin degeneration was not evident in any of the treatment groups in either the cervical or lumbar regions of the spinal cord (Figures 18 and 19). The cells of control ferrets located in the ventral horn of both the cervical and lumbar regions of the spinal cord stained with thionin are shown in Figure 20. A higher magnification of a nerve cell from the cervical spinal cord of a control animal is shown in Figure 21. Nerve cells from the same region but obtained from animals administered 1000 mg TOTP/kg (Figures 22 and 23) showed no detectable qualitative differences when compared to those of the controls (Figures 22 and 23). Although most of the cells appeared normal in 48 FIGURE 12. Longitudinal section of dorsal spinocerebellar tracts in the lumbar region of a spinal cord in a control ferret (300x, Fink-Heimer). FIGURE 13. Longitudinal section of dorsal spinocerebellar tracts in the lumbar region (ME the spinal cord in a control ferret (700x, Fink-Heimer). 49 FIGURE 14. Longitudinal section of dorsal spinocerebellar tracts in the lumbar spinal cord of ferret number F-2 administered 1000 mg TOTP (100X, Fink-Heimer). FIGURE 15. Longitudinal section of dorsal spinocerebellar tracts in the lumbar spinal cord of ferret number F-2 administered 1000 mg TOTP (200x, Fink—Heimer). 50 - - -‘ -t’ ,v...’ FIGURE 16. Longitudinal section of dorsal spinocerebellar tracts of ferret number F-l3 administered 1000 mg TOTP (300x, Fink-Heimer). FIGURE 17. Longitudinal section of dorsal spinocerebellar tracts of ferret number F-13 administered 1000 mg TOTP (600x, Fink-Heimer). 51 FIGURE 18. Cross section of lumbar spinal cord of a control ferret (66X, Speilmeyer). FIGURE 19. Cross section of lumbar spinal cord of a ferret administered 1000 mg TOTP/kg (66X, Speilmeyer). Burn»... 1 l. FIGURE 20. Cross section of cervical spinal cord of a control ferret (87X, Thionin). FIGURE 21. Neuron from ventral IKHHI of cervical spinal cord from a control ferret (700X, Thionin). 53 FIGURE 22. Neuron in the ventral horn of the cervical spinal cord, of a ferret treated with 1000 mg TOTP/kg (700x, Thionin). FIGURE 23. Neuron in cervical spinal cord of a ferret treated with 1000 mg TOTP/kg (700x, Thionin). 54 the treated animal, some swelling of the cells was noticed. The Bioquant area measurements provided evidence that some changes in cell size were present. Neurons located in the lateral part (n: the ventral tunni of the cervical spinal cord in the 1000 mg/kg TOTP-treated ferrets were significantly larger than those of the controls. No significant size difference 'was evident. between cells located. in time medial part (Hi the ventral horn 1J1 1000 mg/kg TOTP-treated animals and controls. The cells located. in the lateral. part. of the 'ventral horn of the lumbar spinal cord in the TOTP-treated animals were not significantly different in size from those of the controls. However, individual animal differences were observed. The nerve cells of ferret F-2 were significantly larger than those from either control animal. Another 1000 mg/kg' TOTP—treated. animal, F—13, did run: statistically' show a significant size difference but did have a smaller calculated mean area than the controls. No significant size differences were found in the area of small neurons located in the medial part of the ventral horn in the lumbar region between treated and control animals. DISCUSSION Three parameters were evaluated in assessing the neurotoxic effects of TOTP on the ferret. These included biochemical changes, functional deficiencies, and 55 histopathological manifestations. The animals on the dermal trial exhibited no statistical significant differences in feed consumption throughout the trial, but several trends were observed. The differences in feed consumption may be attributed to the consistency of the diet, environmental factors, and individual animal differences. The reported average daily feed consumption of 232 g/day for adult male ferrets fed a similar diet by Bleavins and Aulerich (1981) was attained by week 4 for all dermal treatment groups. The ferrets administered dermal doses of 1000 mg TOTP showed the highest feed consumption of any group, which correlates with the increased body weights of the same group during the test. Feed consumption for the animals on the oral trial remained relatively constant throughout the study. The presence of only minor cholinergic effects in orally-treated animals supports the findings of no great increase in feed consumption after cholinergic effects should have diminished (Figure 3). The reason for the apparent trend for increased feed consumption of the control animals in the oral trial during week 2 is unknown. The substantial initial body weight loss for all groups on the dermal trial is consistent with the results obtained from similar studies with chickens and cats (Abou-Donia and Graham, 1979a; Abou—Donia gt a” 1979; Abou-Donia _et a_1., 1983). Other researchers have found that this loss of body 56 weight could be associated with loss of appetite or a direct effect of metabolic processes (Abou-Donia '§t__gl., 1983c). These weight losses were dose dependent and all the animals had gained weight by the termination of the study. The ferrets on the highest dermal dose exhibited the greatest weight gains. This same relationship was noted in the cat by Abou-Donia §t_.gl. (1983b) and has been attributed to the decrease in activity of the highest dose group. The ferrets administered 1000 mg TOTP/kgwdermally also had the greatest feed consumption which could account for the greater weight gain in this group. The early weight loss of the animals on the oral trial was not as marked as for the dermal trial and may be attributed to the lack of progressive neurotoxic clinical signs in the oral as compared to the dermal trial. In this study' the low ‘NTE inhibition (as compared to the Chicken), 46% in the dermally-treated animals and 37% III the orally-treated animals, suggested that cflinical signs of delayed neurotoxicity should not be evident. Johnson (1982) has concluded that 70-75% NTE inhibition is necessary to induce delayed neurotoxic effects in the chicken. However, neurotoxic clinical. and. morphological changes were observed in the ferrets treated with dermal doses of 500 and 1000 mg/kg TOTP. This can be explained through the findings of other investigators who reported that NTE inhibition was not necessarily correlated with the development of delayed 57 neurotoxicity. Bobwhites and mallards were found not to exhibit clinical signs of toxicity even with very high inhibition (66%) of NTE, while chickens which also had high inhibition (70%) of NTE did exhibit signs of neurotoxicity (Bursian e_t E” 1983). Thus, high inhibition of NTE does not always imply delayed neurotoxicity, so low inhibition also might not be related to no development delayed neurotoxic effects. Also, the assay used to determine NTE inhibition must be performed after the time of peak inhibition and before the synthesis of new NTE has begun (Johnson, 1977). Subsequent assays (unpublished data) have shown that the 48 hour post-treatment time interval used in this study was not the best estimate of maximum NTE inhibition in the ferret. Further, because NTE inhibition did not exceed 70-75% in the ferret, this target NTE inhibition may not be a pre-requisite for OPIDN. The severity' of the neurological dysfunctions observed in the treated ferrets was directly related to dose and route of exposure. Although the evaluation of the observed clinical signs of delayed neurotoxicity was subjective, the results of such evaluations have been found to be very sensitive in detecting early onset of neurologic deficits (Sprague §t_gl., 1980). All animals exhibiting signs of OPIDN followed the same progressive sequence. Initial leg weakness was observed on the average of 16 days post-treatment, with 11 58 days being the earliest an animal displayed neurotoxic signs. These findings are consistent with the results reported by Abou-Donia gt 31. (19830) for cats which indicated that initial delayed neurotoxic signs were present two to three weeks after administration of TOTP. Leg weakness was followed by the progressive onset of other dysfunctions that included ataxia, uncoordinated movements, high stepping gait, sliding walk, and partial paralysis. These progressive clinical signs closely followed the pattern of OPIDN in cats (Abou-Donia et_gl., 1983b). The advanced. neurological examinations yielded. abnormal responses only after the ferrets received. an R3 .rating in the clinical observations (Tables 6 and 7). This is consistent with other evidence that postural reactions and spinal cord reflexes remain normal during the early onset of delayed neurotoxicity (Abou-Donia e_t 31., 1983b). During the early ataxia stages (R3), abnormal extensor thrust reflexes were noted indicating defects in sciatic and tibial nerve functions. Only at the severe ataxia state (R4) were substantial abnormal postural reactions observed, suggesting lesions in the spinal cord. A clinical examination rating of R5, indicating partial paresis corresponded with high scores in all neurological tests, except for the wheelborrow test. At this stage, severe deficits in neurologic responses were observed, with no consequential effect on forelimb functions. This dominant 59 effect on hindlimbs as opposed to frontlimbs is a characteristic of OPIDN (Abou-Donia gt _a_l_., 1983b; Abou-Donia et; 21,, 1983c). The most characteristic abnormal response of the ferret was demonstrated in the hanging/grasping neurological test. Ferrets at the highest dose level displayed a progressive abnormal response best described as the lack of spreading out of the hindlimbs and complete extraction of the claws when held ixlaa hanging position. This response was also not observed until a general observation rating of R3 was obtained, implying possible spinal cord lesions. The lack of jprogressive clinical signs in time ferrets on the oral trial is not in complete agreement with the results obtained from other investigators. Although studies have shown the acute toxicity of dermally-applied TOTP to be twice that of oral administration, progresSive signs were still apparent in orally-treated animals (Abou-Donia et_gl,, 1983c). However, these clinical signs 'were not as severe: as those found in the animals on the dermal trial and also were not as prominent by the end. of the trial (Abou-Donia qgt.:gl., 1974). The ferrets on the oral trial displayed clinical signs of recovery towards the end of the study, whereas the dermally treated animals did not. The cat also has been found to exhibit signs of recovery (Abou-Donia gt 31., 1983b). The lesions present in the lumbar spinal cord of ferrets treated dermally with 1000 mg TOTP/kg are indicative of the 60 observed clinical signs. The dorsal spinocerebellar tracts have been. demonstrated to ‘terminate only' in 'the region. of the cerebellum that controls hindlimb functions and these tracts mediate impulses from proprioreceptors (Brodal, 1975). These proprioreceptors convey information tx> the tracts from muscle spindles, tendon organs, and pressure receptors. Some dorsal spinocerebellar units also convey impulses from flexor reflex afferents which control the .flexor reflex in animals. Thus, the clinical dsyfunctions observed in the TOTP-treated ferrets can be histopathologically explained by lesions in these areas, especially the neurological deficits seen i11 flexor aumi extensor responses. Although others have demonstrated lesions characteristic of OPIDN in ventral—descending tracts III the lumbar anui thoracic regions and also iri‘the dorsal-lateral tracts (Hf the cervical region (Abou-Donia and Graham, 1979; Abou-Donia §1_ 21,, 1983b), these lesions were not noted in the TOTP-treated ferrets. The ventral spinocerebellar tracts are also concerned.‘with the transmission of impulses from the hindlimbs, but the fibers are much thinner than those located in the dorsal region (Brodal, 1975). Conclusions have been drawn suggesting that axon degeneration in OPIDN is most often associated with larger diameter axons (Baron, 1981). This may explain why lesions were not detected in the ventral regions. Further, axon degeneration was more extensive in animal F-l3 (Figures 61 l4 and 15) which also exhibited more severe clinical signs, than animal F-2 (Figures 12 and 13) which was also treated dermally with 1000 mg/kg TOTP. This suggests a correlation between severity of clinical signs and histopathologic changes. Abou—Donia g _a_1_. (1983b) described histopathological changes as a function of size of the dose, frequency and duration of the administration, severity of clinical signs, length of the period between the onset and termination of ataxia, and the site of tissue sampling. The evidence obtained from the histopathologic evaluations of the ventral motor neurons also substantiates the clinical results. Although qualitative changes were not easily detectable, area measurements suggested that changes in cell size had occurred in the TOTP—treated ferrets. Differences were only found in neurons in the lateral part of the ventral horn of the cervical spinal cord and in the larger, dorsally—located cells of the lumbar ventral horn. Anatomical investigations have shown that the medial cells supply muscles of the neck and trunk, while the lateral neurons supply the muscles of limbs (Brodal, 1975). Cells associated with functions of the distal extremities are found in the most dorsal regions of the ventral horn. The cells in the cervical spinal cord were significantly larger in the 1000 mg TOTP/kg dermally—treated animals when compared to the controls. The cells in the lumbar region were variable as the cells 62 of animal F—2 were significantly larger than controls, but the cells from animal F—13 were significantly smaller. Although statistics conducted on a small size sample are not that reliable, these results suggest that the animal, F-l3, exhibiting the most severe clinical signs and axon degeneration may also have shown initial stages of neuronal atrophy subsequent to swelling of the cells. Animal F-2 exhibited signs of neuron swelling but had not yet advanced to the atrophy stage. There are conflicting reports concerning on the effect of OPIDN on mOtor neurons. Some reports indicate that the cell bodies are not involved. in the toxic process (Cavanagh and Koller, 1979; Cavanagh, 1973). Ahmed and Glees (1971), however, concluded from their observations in the slow loris that chromalytic neurons were present in the ventral horns of the lumbar regions. These neurons were characterized by an eccentric nucleus, peripheral arrangement of nissl substance, and atrophy or disintegration of neurons. Many studies have used various staining methods to demonstrate demyelination in OPIDN. In. the present study, the stained myelin tissues did not reveal conclusive evidence that demyelination had occurred. Lack of myelin staining was found in both control and treated animals. Therefore, detection of myelin loss due to the administration of TOTP could run: be determined. Cavanagh (1973) stated that myelin stains are often unsatisfactory and misleading because only 63 in cases of advanced degeneration do myelin stains show appreciable differences. From results obtained in this study, it was concluded that dermal administration of TOTP was more effective than oral administration 111 producing' delayed. neurotoxicity. Abou-Donia .gt__g1. (1983b) have related this difference in toxicity in route of administration in the cat to slow absorption of chemicals from the gastrointestinal tract and variable absorption in individual orally-treated animals. The recovery observed in the ferrets administered TOTP via the oral route may be attributed to peripheral nerve regeneration (Abou-Donia e1_.g1., 1983c). Peripheral nerve damage occurs earlier in the OPIDN sequence and can be reversible, but spinal cord lesions are irreversible (Abou-Donia and Ckaham, 1979b). Thus, the neurotoxic effect and recovery may be hypothesized to be dependent upon administered dose and route of exposure. The ferret's sensitivity to OPIDN differed slightly from both the chicken and the cat. Although the ferret was demonstrated not tx>lx3 as sensitive to OPIDN as the chicken, similarities exist in that no recovery in the dermally-treated animals was observed. The ferret also had similar responses to time cat 1J1 the neurological examinations but IN) recovery was observed in dermally-treated ferrets as has been reported for cats. The ease of handling and caring for the ferret, 64 along with the species sensitivity to delayed neurotoxicity, make this species an attractive future alternative for OPIDN testing. However, further research, including long-term chronic trials, sequencial investigations of histopathological changes, and studies in distribution, metabolism, and elimination of delayed neurotoxins should be conducted before an accurate assessment of the use of the ferret as a model species can be made. CONCLUSIONS Based upon the results of this study to assess the sensitivity of the ferret to OPIDN, the following conclusions were drawn. 1. Ferrets are a sensitive species to OPIDN when administered dermal doses of TOTP and are less sensitive when administered oral doses. 2. Clinical signs of delayed neurotoxicity were observed 16 days post-treatment and were characterized by ataxia that progressed to partial paralysis of the hindlimbs. 3. Histopathological. lesions .included anxnm degeneration in the dorsal spinocerebellar tracts of the lumbar spinal cord and changes in cell size of the ventral horn motor neurons. 4. Onset of delayed neurotoxicity and recovery was dependent upon dose and route of exposure. Recovery was only observed in animals administered TOTP via the oral route. 5. Clinical and histopathological symptoms of OPIDN were present even at minimal levels of NTE inhibition. 6. The ferret was not as sensitive as the chicken to OPIDN’ but similar in sensitivity' to tflma cat. The ferret appears to be a satisfactory species for testing the delayed neurotoxic potential of chemicals, but further research on OPIDN with the ferret should be conducted. 65 REFERENCES Abou—Donia, M.B., 1981. Organophosphorus ester-induced delayed neurotoxicity. Ann. Rev. Pharmacol. Toxicol. 21:511-548. Abou-Donia, M.B., G. Doyle, H.M. Graham, and K.M. Abdo, 1983a. 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