Biochemical and neuropathological assessment of triphenyl phosphite in rats

The putative neurotoxicity of the organophosphorus compound triphenyl phosphite (TPP) was examined in Long Evans, adult male rats. Animals were exposed to two 1.0 ml/kg (1184 mg/kg) injections (sc) of TPP spaced 1 week apart and sampled for biochemical and neuropathological examination. At the time of sampling, rats displayed dysfunctional changes including tail rigidity, circling, and hindlimb paralysis. Neuropathic damage was confined to the lateral and ventral columns of all spinal levels and consisted of myelin ellipsoids and giant axonal swellings filled with smooth endoplasmic reticulum. Wallerian-like degeneration was observed in the spinal roots, the sciatic nerve, and tibial branches. Biochemical assessment of brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activity was determined 1, 4, 24, 48, and 72 hr after the second TPP treatment. Both enzyme activity concentrations were depressed maximally at 48 hr postexposure by 30 and 39%, respectively. Serum cholinesterase, sampled 48 hr after the second TPP exposure was depressed by 33%. Data from this study indicate that subchronic exposure to the organophosphite TPP results in severe neurotoxic consequences which differ from those previously described in rats with organophosphorus-induced delayed neuropathy.     

Time-dependent changes of lipid peroxidation and antioxidative status in nerve tissues of hens treated with tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP) could induce a delayed neurodegenerative condition known as organophosphorus easter-induced delayed neurotoxicity (OPIDN) in human beings and sensitive animals. However, the mechanisms of OPIDN remain unknown. This study investigated the time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues for elucidating the mechanism of OPIDN induced by TOCP. Adult hens were treated with TOCP by gavage at a single dosage of 750 mg/kg. TOCP was dissolved in corn oil and administered at 0.65 ml/kg. The control hens received an equivalent volume of corn oil by gavage. Hens were sacrificed after 0, 5, 10, 15 and 21 days of treatment and the cerebrum, spinal cord, sciatic nerve were dissected, homogenized and used for the determination of lipid peroxidation and antioxidative status. The results showed that treatment with TOCP increased lipid peroxidation and reduced the antioxidative status in cerebrum, spinal cord and sciatic nerve. The levels of MDA increased by 33% (P<0.01) in cerebrum on 5th day after TOCP treatment and at clinical sign score of 1-2, and increased respectively by 32% and 15% (P<0.01) in spinal cord and sciatic nerve on 10th day after TOCP treatment and at clinical sign score of 3-4. Further changes of MDA were also observed after 15 and 21 days post-dosing and at clinical sign score of 5-6 and 7-8. There is a decrease in the activities of SOD, GSH-Px, GR, anti-ROS, and GSH content in cerebrum, spinal cord and sciatic nerve of hens after 5, 10, 15 and 21 days post-dosing and at clinical sign score of 1-2, 3-4, 5-6 and 7-8. Thus, OPIDN induced by TOCP was associated with elevation of lipid peroxidation and reduction of antioxidative status, and the time-dependent changes of these indexes in hens nerve tissues occurred. Sciatic nerve was the main target tissue and MDA was most sensitive among all indexes. The time-dependent and tissue specific changes of lipid peroxidation and antioxidative status in cerebrum, spinal cord and sciatic nerve suggest that ROS and concomitant lipid peroxidation, at least in part, are involved in the toxic effects of TOCP on nerve tissues and that oxidative stress may play a role in the occurrence and development of OPIDN induced by TOCP.     

Delayed neuropathy and acute toxicity studies with pirimiphos-methyl in the hen

This paper describes studies aimed at determining the acute anticholinergic and delayed neurotoxic potential of the organophosphate insecticide pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate) in the hen. Delayed neuropathy was assessed by biochemical measurement of neuropathy target esterase (NTE) activities in the brain and spinal cord, clinical signs of neuropathy over two 21-day periods and histological assessment of nervous tissue. Acetylcholinesterase (AChE) activity was also determined in the brain and spinal cord. Hens were given a single oral dose of 100 mg kg-1 pirimiphos-methyl, which was followed by a repeated dose after 21 days. Tri-o-cresyl phosphate (TOCP), 500 mg kg-1, was used as a positive control. All pirimiphos-methyl-treated hens received prophylactic doses of N-methylpyridinium-2-aldoxime methanesulphonate (P2S) and atropine sulphate. Hens dosed with pirimiphos-methyl had very low AChE activities (less than 20% of control) in both the brain and spinal cord, 24 and 48 h after dosing. In the TOCP-treated hens, the activities were about 90% of control. NTE activities in the brain and spinal cord of pirimiphos-methyl-treated hens were identical to those in the controls, while they were profoundly inhibited (greater than 80%) in the TOCP-treated hens. All hens dosed with pirimiphos-methyl showed the expected signs of AChE inhibition and, following recovery, usually by Day 5, no clinical signs of delayed neuropathy were seen. The TOCP-treated hens developed clinical signs of neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

An assessment of the neurotoxic potential of fenitrothion in the hen

The potential of single, toxic doses of fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl)phosphorothioate) to elicit delayed neurotoxicity in the adult White Leghorn hen was compared to the effects produced following similar treatment with the known neurotoxin, tri-o-tolyl phosphate (TOTP). Hens (2.0-2.5 kg body wt) received single oral doses of fenitrothion (500 mg/kg) or TOTP (500 mg/kg), the resulting toxicity being assessed by measuring biochemical (brain and spinal cord acetylcholinesterase (AChE) and neurotoxic esterase (NTE), physiological (motor function) and morphological (cross- and longitudinally-sectioned and stained preparations) parameters of the brains, spinal cords and sciatic nerves of groups (n = 5) of hens at 24 h, 7, 14, 28, 42 and 56 days post-treatment. At 24 h after treatment, fenitrothion caused a marked inhibition of neuronal AChE while TOTP had no effect. In contrast, TOTP caused a significant inhibition of NTE whereas fenitrothion was without effect. At 7 days after treatment, the NTE was still significantly reduced in TOTP-treated hens but normal levels of activity were detected at 14 days post-treatment. No alternation in NTE activity was found in any fenitrothion-treated hens. A characteristic, central-peripheral, distal axonopathy was observed following treatment with TOTP, mild signs appeared 7-14 days post-treatment and increased in severity up to 28 days after treatment, concomitant with morphological changes primarily in the sciatic nerves and spinal cords. Minimal morphological changes were elicited by fenitrothion at this dosage, the tissues appearing no different than those seen in vehicle-treated control hens. The results demonstrated that fenitrothion was distinctly different from TOTP in the biochemical, physiological and morphological effects produced in acutely treated hens and that fenitrothion could not be considered to be neuropathic in the classical manner of TOTP.     

Triphenylphosphite neuropathy in hens

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5–10 versus 7–14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10–15 versus 4–6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7–12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10–120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10–30 mg/kg s.c.) This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.     

Antibodies to neural proteins in organophosphorus-induced delayed neuropathy (OPIDN) and its amelioration

The development of OPIDN and the efficacy of experimental intervention using the calcium-channel blocker verapamil were used as a model to test the serial time-measurements of serum autoantibodies against neuronal cytoskeletal proteins [e.g., neurofilament triplet (NF)] and glial proteins [myelin-basic protein (MBP) and glial fibrillary-acidic protein (GFAP)] as biomarkers of neurotoxicity and its amelioration. Ten White Leghorn hens (>7 months, 1.2-1.8 kg) were administered phenyl-saligenin phosphate (PSP; 2.5 mg/kg; im), a dose reported to induce a 70% decrease in neurotoxic esterase (NTE) activity. Five of the hens were administered verapamil (7 mg/kg; im) for 4 days starting one day before PSP administration. Serum was isolated from blood collected by serial brachial venepuncture before PSP (day 0) administration and on days 3, 7 and 21 after PSP administration, each hen acting as its own control. Serum antibodies (IgG) to NF-L, NF-M, NF-H, MBP, and GFAP were assayed using an ELISA. There were no detectable levels of antibodies on days 0 and 3. IgG against all neural proteins were detected on days 7 and 21, with titer levels being significantly (p< or =0.05) higher in sera of hens receiving PSP only. Anti-NF-L titers were highest compared to those against NF-M, NF-H or MBP at 21 days. Titers of anti-NF-L and anti-MBP significantly (p< or =0.01) correlated with clinical scores at days 7 and 21. Detection of anti-NF and anti-MBP antibodies confirms the neuroaxonal degeneration accompanied by myelin loss reported in this model of OPIDN and the amelioration of neuropathy using verapamil. The detection of anti-GFAP antibodies suggests CNS involvement in OPIDN, since astrocytes are only found therein. This study demonstrates that detection of neuroantibodies can be used as biomarkers of neuropathy development and to monitor the amelioration resulting from therapeutic intervention. Together with biomarkers of exposure neuroantibodies can be used to monitor neuropathogenesis due to environmental or occupational exposures.     

Organophosphorus compound-induced delayed neurotoxicity in white leghorn hens assessed by Fluoro-Jade

Certain organophosphorus (OP) compounds can induce a delayed neuropathy, termed OPIDN, that involves central and peripheral nervous system axons, terminals, and perikarya. Historically, OPIDN has been characterized by staining neural sections with silver or hematoxylin and eosin (H and E). This study utilized a novel staining method, Fluoro-Jade, for evaluating the distribution and extent of OPIDN in the central nervous system of hens. Results were then compared to synoptically sectioned and stained H and E preparations. White Leghorn hens were injected with phenyl saligenin phosphate (PSP, 2.5 mg/kg, intramuscular [im]), triphenyl phosphite (TPPi, 500 mg/kg, subcutaneous [sc]), or dimethyl sulfoxide vehicle (DMSO, 0.5 ml/kg, im or sc) and evaluated clinically for signs of neurological dysfunction associated with OPIDN. Hens were sacrificed 7, 14, and 21 days post dosing. Brains and spinal cords were removed immediately following sacrifice, fixed in formalin, and embedded in paraffin. Microtome-cut sections (7 micro m) were then stained with Fluoro-Jade (0.001%, w/v) or H&E. Staining with Fluoro-Jade revealed time-dependent degeneration of nerve fibers and terminals (with PSP and TPPi), or cell bodies (with TPPi) in lamina VII, spinocerebellar, and medial pontine-spinal tracts of the lumbar spinal cord, in white matter and mossy fibers of foliae I-V and IX of the cerebellum, and in medullary, pontine, and midbrain nuclei and paleostriatal fibers surrounding the optic tract. TPPi-induced degeneration was more extensive than that induced by PSP and affected additional cerebellar folia, medullary, pontine, midbrain, and forebrain nuclei and fiber tracts. H&E-stained sections revealed fewer sites of neurodegeneration when compared to Fluoro-Jade. These results demonstrate that Fluoro-Jade is a sensitive method for staining neural tissue affected by OPIDN.     

Delayed neurotoxicity of triphenyl phosphite in hens: Pharmacokinetic and biochemical studies

The organophosphorus compound, triphenyl phosphite (TPP), caused ataxia in chickens 8-14 days after single po or iv administration. The po and iv ED50 values were 1414 and 35.4 mg/kg, respectively. Chickens which developed ataxia lost 14.4 +/- 2.5% (mean +/- SEM, n = 14) of their initial weight at 28 days and the paralyzed birds showed a severe reduction of 29.3 +/- 2.9% (n = 13) of their initial weight at death or at 28 days after dosing. For the first 4-hr interval after iv injection of 50 mg/kg, the elimination of TPP from plasma consisted of at least two exponential phases; the half-lives of the first and second phases were approximately 30 and 60 min, respectively. When the birds received 100 mg/kg (iv) fatty tissue showed the highest TPP concentration, e.g., 215 micrograms/g fresh wt at 6 hr postdosing. The half-life was approximately 24 hr. Among neural tissues, the sciatic nerve had the highest concentration, followed by the spinal cord, the cerebellum, and the cerebrum. The red muscles, such as adductor magnus, contained about 4-30 times as much TPP as did the white muscles, such as biceps brachii, 6 hr after treatment. Time course effects of TPP treatment on mitochondrial enzymes in leg skeletal muscles were examined by treating hens with 50 mg/kg (iv) and euthanizing the birds at 6 hr to 8 days postdosing. The creatine kinase (CK) activities of the adductor and the soleus were significantly decreased at 2 (48 hr), 4, and 8 days, and at 4 and 8 days postdosing, respectively. Adductor magnus and soleus succinate dehydrogenase (SDH) activities were decreased markedly at 24 and 48 hr, and at 2 (48 hr), 4, and 8 days, respectively. Cytochrome oxidase (COD) activity in adductor magnus and soleus did not decrease during the time course. Biceps femoris CK, SDH, and COD activities were not affected by TPP treatment at this dosage. These results suggest that TPP administration affects the mitochondrial metabolism in skeletal muscle, especially red muscle of chickens.     

Use of the Biventer Cervicis Nerve-Muscle Preparation to Detect Early Changes following Exposure to Organophosphates Inducing Delayed Neuropathy

Use of the Biventer Cervicis Nerve-Muscle Preparation to DetectEarly Changes following Exposure to Organophosphates InducingDelayed Neuropathy. EL-FAWAL, H. A. N., JORTNER, B. S., ANDEHRICH, M.(l990). Fundam. Appl. Toxicol. 15, 108–120.Indices of organophosphorus (OP)-induced delayed neuropathy(OPIDN) in the hen model have traditionally been restrictedto the early inhibition of neuropathy target esterase (NTE)and ataxia with associated pathological changes in hind limbperipheral nerve which occur more than 7 days after OP exposure.The biventer cervicis nerve-muscle preparation was used to evaluateOPIDN in adult hens at various time periods after treatmentwith either the protoxicant tri-o-tolyl phosphate (TOTP), 360mg/kg po, or the active congener phenyl saligenin phosphate(PSP), 2.5 mg/kg im. NTE activity was 21 and 48% of controlfor TOTP and PSP, respectively, 4 days after administration.Clinical signs were notable by 10 days and progressed in severityto paralysis by 21 days. Partial clinical recovery was evidentat 37 days. Denervation hypersensitivity of biventer cervicismuscle to acetylcholine (ACh) was evident as early as 4 daysfollowing TOTP or PSP treatment. The sensitivity to ACh wasgreatest 21 days after OP administration, with partial recoveryat 37 days. Strength-duration curves (SDC) of preparations fromOP-treated hens showed an increase in excitability thresholdsand elevated rheobase with shorter chronaxie than did preparationsfrom controls as early as 4 days following treatment with eithercompound. SDC at 37 days indicated partial reinnervation. Peripheralnerve myelinated fiber degeneration and regeneration consistentwith these physiological changes was seen on histopathologicalexamination. This study suggests that the biventer cervicisnerve-muscle preparation may prove useful for detection of functionaland morphological changes that occur during the interval betweenNTE inhibition and appearance of clinical deficits.     

Age-Related Effects of Triphenyl Phosphite-Induced Delayed Neuropathy on Central Visual Pathways in the European Ferret (Mustela putorius furo)

The objective of this study was to investigate the relationshipbetween the maturation of visual system neurons and the onsetof their susceptibility to triphenyl phosphite (TPP)-induceddelayed neurotoxicity in the European ferret. We administeredsingle subcutaneous doses of TPP (1184 mg/kg body wt) to 1-to 10-week-old ferret kits to assess the effects on connectionsand neurons of the developing lateral geniculate thalamic nucleus(LGN) and primary visual cortex. Brains were processed witha modified Fink-Heimer silver-impregnation method. Axonal andterminal degeneration were first noted in the LGN of kits injectedat 5 weeks of age. The severity of the degeneration increasedin kits injected at later ages and reached adult densities andconfigurations in ferrets injected at 10 weeks of age. Degeneratingneuronal cell bodies were also present in the LGN of kits injectedat 7 weeks of age and older. In the visual cortex, axonal andterminal degeneration were consistently present in kits injectedat 8 weeks of age and attained adult-like densities in kitsinjected at 10 weeks of age. Previous studies have reportedthat the ferret visual system appears to reach anatomical maturity(as defined by mature LGN lamination patterns, the locationand density of axon terminals originating from neurons in theretina and LGN, and the migration and synaptic connections ofcortical neurons) by 4–5 weeks of age. A temporal comparisonof these normal developmental data with the degeneration dataobtained in the present study suggests that immature neuronsin the visual system of the ferret are not susceptible to TPP-in-duceddelayed neurotoxicity but only become so after they have achievedsome degree of maturity. Whether the LGN neurons undergoingdegeneration are directly affected by TPP or are showing a transneuronalresponse to loss of afferent input remains Unresolved.     

The relationship between neurological damage and neurotoxic esterase inhibition in rats acutely exposed to tri-ortho-cresyl phosphate

A rodent model of organophosphorus-induced delayed neuropathy (OPIDN) has been developed using Long-Evans adult male rats exposed to tri-ortho-cresyl phosphate (TOCP). In the present study an attempt was made to relate neurochemical with neuropathological changes in rats exposed to single dosages of TOCP ranging from 145 to 3480 mg/kg. The degree of neurotoxic esterase (NTE) inhibition, measured at 20 and 44 hr and at 14 days postexposure was correlated with the appearance of spinal cord pathology 14 days postexposure in a separate group of similarly dosed rats. Those dosages that inhibited mean NTE activity in spinal cord greater than or equal to 72% and brain greater than or equal to 66% of control values within 44 hr postexposure produced marked spinal cord pathology 14 days postexposure in greater than or equal to 90% of similarly dosed animals. In contrast, dosages of TOCP which inhibited mean NTE activity in the spinal cord less than or equal to 65% and in the brain less than or equal to 57% produced spinal cord pathology in less than or equal to 15% of the animals. These data indicate that NTE inhibition may be used as a biochemical predictor for TOCP-induced neurological damage in rats.     

Changes of mitochondrial ultrastructures and function in central nervous tissue of hens treated with tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, is capable of producing organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animals. The mechanism of OPIDN has not been fully understood. The present study has been designed to evaluate the role of mitochondrial dysfunctions in the development of OPIDN. Adult hens were treated with 750 mg/kg·bw TOCP by gavage and control hens were given an equivalent volume of corn oil. On day 1, 5, 15, 21 post-dosing, respectively, hens were anesthetized by intraperitoneal injection of sodium pentobarbital and perfused with 4% paraformaldehyde. The cerebral cortex cinerea and the ventral horn of lumbar spinal cord were dissected for electron microscopy. Another batch of hens were randomly divided into three experimental groups and control group. Hens in experimental groups were, respectively, given 185, 375, 750 mg/kg·bw TOCP orally and control group received solvent. After 1, 5, 15, 21 days of administration, they were sacrificed and the cerebrum and spinal cord dissected for the determination of the mitochondrial permeability transition (MPT), membrane potential (Δψ(m)) and the activity of succinate dehydrogenase. Structural changes of mitochondria were observed in hens' nervous tissues, including vacuolation and fission, which increased with time post-dosing. MPT was increased in both the cerebrum and spinal cord, with the most noticeable increase in the spinal cord. Δψ(m) was decreased in both the cerebrum and spinal cord, although there was no significant difference in the three treated groups and control group. The activity of mitochondrial succinate dehydrogenase assayed by methyl thiazolyl tetrazolium (MTT) reduction also confirmed mitochondrial dysfunctions following development of OPIDN. The results suggested mitochondrial dysfunction might partly account for the development of OPIDN induced by TOCP.     

Triphenyl phosphite: in vivo and in vitro inhibition of rat neurotoxic esterase

Organophosphorus compounds which, after acute administration, inhibit neurotoxic esterase (NTE) by greater than or equal to 65% and undergo a subsequent "aging" reaction, produce a delayed neuropathy characterized by degeneration of large and long nerve fibers (OPIDN). The present studies examine in detail the NTE-inhibiting properties of triphenyl phosphite (TPP), a plasticizer which produces ataxia and degeneration of the spinal cord in animals. A neurotoxic dosing regimen (1184 mg/kg/week, sc, for 2 weeks) inhibited both brain and spinal cord NTE (less than or equal to 40%) only marginally 4 and 48 hr postdosing. By contrast, TPP was shown in vitro to be a potent (150 = 0.98 microM) inhibitor of rat brain NTE relative to Mipafox or diisopropyl phosphorofluoridate. Compounds structurally related to TPP (i.e., triphenyl phosphate, triphenyl phosphine, trimethyl phosphite, and phenol) failed to inhibit NTE in vitro at less than 10 microM concentrations. Close examination of the TPP inhibition of NTE showed a nonlinear relationship between the duration of incubation time and loss of log(NTE activity). Preincubation of 10 microM TPP in buffer (37 degrees C) resulted in a time-dependent loss of TPP's ability to inhibit NTE. In summary, TPP is a powerful NTE inhibitor in vitro, but only a marginal NTE inhibitor after in vivo administration. These results raise questions as to the causal events mediating TPP-induced neuropathy in the rat.     

Delayed neurotoxicity of leptophos and related compounds: differential effects of subchronic oral administration of pure, technical grade and degradation products on the hen

Pure and technical grade leptophos (O-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonothioate) and some of its degradation products were screened for delayed neurotoxicity following daily oral administration to hens. Minor modification of the leptophos molecule did not abolish its neurotoxic effect. In contrast, the hydrolytic products, 4-bromo-2,5-dichlorophenol and phenylphosphonic acid, had no neurotoxic action. The effectiveness of esters to induce delayed neurotoxicity was, in descending order: O-2,5-dichlorophenyl O-methyl phenylphosphonothioate (desbromoleptophos) >; pure leptophos > technical grade leptophos > O-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonate (leptophos oxon). Leptophos oxon was only weakly effective as a neurotoxic agent probably because it was unstable in the body and by the time it had reached the target for neurotoxic action it had been hydrolyzed to nonneurotoxic products. Histopathologic examination of hens that died during the study or were killed 30 days after completion of treatment showed marked axon and myelin degeneration in the sciatic, peroneal, and tibial nerves and spinal cord of most hens. The lesions in peripheral nerves were generally seen earlier and in fewer hens than those in spinal cord. The most consistent histopathological changes were degeneration of axons and myelin in the spinal cord. Leptophos oxon was the most potent inhibitor of brain and plasma cholinesterases. Also, the activity of plasma butyryl cholinesterase was more inhibited than brain acetylcholinesterase. Controls consisted of four groups of hens given daily oral doses of 10 mg/kg tri-o-cresyl phosphate (TOCP), 1.0 mg/kg O,O-diethyl-O-4-nitrophenyl phosphorothioate (parathion), and an empty gelatin capsule. TOCP-treated hens developed delayed neurotoxicity while those given parathion showed initial leg weakness but subsequently recovered without developing delayed neurotoxicity. Control hens receiving gelatin capsules remained normal. This study suggests that changes in structure of leptophos as it is absorbed, metabolized, accumulated, and eliminated in vivo, significantly affect the development and severity of delayed neurotoxicity. The chicken continues to be an ideal model for screening organophosphorus pesticides for their capacity to produce delayed neurotoxicity.     

Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: a study of molecular, cellular, and anatomical parameters

Organophosphorus-ester induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral, distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken that results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 ml/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were euthanized at different time points such as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum, brainstem and spinal cord were quickly dissected and frozen for mRNA (northern) studies. Northern blots were probed with BCL2, GADD45, beta actin, and 28S RNA to investigate their expression pattern. Another set of hens was treated for a series of time points and perfused with phosphate buffered saline and fixative for histological studies. Various staining protocols such as Hematoxylin and Eosin (H&E); Sevier-Munger; Cresyl echt Violet for Nissl substance; and Gallocynin stain for Nissl granules were used to assess various patterns of cell death and degenerative changes. Complex cell death mechanisms may be involved in the neuronal and axonal degeneration. These data indicate altered and differential mRNA expressions of BCL2 (anti apoptotic gene) and GADD45 (DNA damage inducible gene) in various tissues. Increased cell death and other degenerative changes noted in the susceptible regions (spinal cord and cerebellum) than the resistant region (cerebrum), may indicate complex molecular pathways via altered BCL2 and GADD45 gene expression, causing the homeostatic imbalance between cell survival and cell death mechanisms. Semi quantitative analysis revealed that the order of severity of damage declines from the spino-cerebellar, ventral, and dorsal tract respectively, suggesting neuroanatomical specificity. Thus, early activation of cell death and cell survival processes may play significant role in the clinical progression and syndromic clinical feature presentation of OPIDN.     

Brain acetylcholinesterase, acid phosphatase, and 2',3'-cyclic nucleotide-3'-phosphohydrolase and plasma butyrylcholinesterase activities in hens treated with a single dermal neurotoxic dose of S,S,S-tri-n-butyl phosphorotrithioate

The changes in brain acetylcholinesterase (AChE), acid phosphatase (APase), and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP), and plasma butyrylcholinesterase (BuChE) activities were investigated in hens treated with a single, dermal dose (100-1000 mg/kg) of S,S,S-tri-n-butyl phosphorotrithioate (DEF). Three control groups consisted of hens left untreated, given a single, dermal dose of 500 mg/kg tri-o-cresyl phosphate (TOCP, positive control for organophophorous compound-induced delayed neurotoxicity), or 10 mg/kg O,O-diethyl O-4-nitrophenyl phosphorothioate (parathion, negative control). Brain AChE activity, determined 28 days after application, was significantly inhibited in hens given 500-1,000 mg/kg DEF and in TOCP- and parathion-treated hens. In contrast, brain APase and CNP activities were significantly higher in all treatments as compared with those of the untreated hens. Parathion, however, caused the least increase in these enzymatic activities as compared to DEF or TOCP. A single, dermal dose of DEF or TOCP also caused an initial decrease in plasma BuChE activity with maximum depression of enzymatic activity observed 1 to 7 days after administration. This decrease was dose dependent and the enzymatic activity showed partial recovery with time. Hens treated with single, dermal doses of DEF, ranging from 250 to 1000 mg/kg, developed ataxia which progressed to paralysis in some hens. Histopathologic examination revealed axon and myelin degeneration of the spinal cord and peripheral nerves of some hens. The severity and frequency of the neuropathologic lesions were dose dependent. Neurologic dysfunctions and neuropathologic lesions seen in DEF-treated hens were similar to those exhibited in TOCP-treated hens. While parathion produced acute cholinergic effects, it did not cause delayed neurotoxicity. The changes in brain and plasma enzymes are discussed in relation to their role in the pathogenesis of DEF-induced delayed neurotoxicity.     

Inhaled iron, unlike manganese, is not transported to the rat brain via the olfactory pathway

Iron and manganese share structural, biochemical, and physiological similarities. The objective of this study was to determine whether iron, like manganese, is transported to the rat brain via the olfactory tract following inhalation exposure. Eight-week-old male CD rats were exposed to approximately 0.31 mg Fe per m(3) (mass median aerodynamic diameter = 2.99 microm; geometric standard deviation = 1.15) via inhalation for a target duration of 90 min. Following exposure, rats were euthanized immediately (0) or at 1, 2, 4, 8, or 21 days postexposure. In addition to nasal and regional brain tissues, blood, and viscera were also collected. 59Fe concentrations were determined by gamma spectrometry. Further, heads were collected and frozen, and autoradiograms were prepared to visualize the location of 59Fe from the nose to the brain. Finally, olfactory mucosa samples collected at 0, 2, 4, and 21 days postexposure were further analyzed using high-performance liquid chromatography (HPLC) plus gamma spectroscopy to determine the association between 59Fe and transferrin. Data obtained from gamma spectrometry revealed that most of the iron remained in the nasal regions of the olfactory system and that less than 4% of iron deposited on the olfactory mucosa was observed in the olfactory bulb. Autoradiograms confirmed the data obtained from gamma spectrometry. 59Fe activity was absent in the olfactory regions of the brain even 4 days postexposure. Further, HPLC-gamma spectroscopy analyses indicated that 59Fe in the olfactory mucosa was coeluted with transferrin. Hence iron, unlike manganese, is not readily transported to the brain via the olfactory tract.     

Delayed neurotoxicity from long-term low-level topical administration of leptophos to the comb of hens.

The production of delayed neurotoxicity in hens following percutaneous administration of leptophos [O-(4-bromo-2,5-dichlorophenyl) O-methyl phenylphosphonothioate] has been investigated. By applying a solution of the insecticide in acetone to the comb, seven groups of three laying hens were given daily a single percutaneous dose of 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, or 20.0 mg/kg of leptophos for 183–323 days. All hens given 0.5–20 mg/kg developed ataxia. The group of hens given a daily dose of 0.1 mg/kg of leptophos showed no abnormalities in gait or behavior. The severity of the clinical condition depended on the size of the daily applied dose. The “latent period” and “total administered dose” before onset of ataxia depended on the daily topically applied dose. The most consistent histopathological changes were degeneration of myelin and axons in spinal cords of intoxicated birds. The severity of change was greatest in hens receiving the highest doses. While plasma cholinesterase was inhibited in all treated birds, plasma acid phosphatase activity was significantly increased. The present investigation shows that long-term low-dose applicaton of leptophos to the comb produced delayed neurotoxicity in hens similar to that reported for the oral administration of this compound.