Neuropathological studies on cycloate-induced neuronal cell death in the rat brain.

The herbicide cycloate (carbamothioic acid, ethyl(cyclohexyl)-S-ethyl ester) given as a single oral dose to rats, caused selective neuronal cell death in two regions in the rat forebrain, the pyramidal neurons of layers II-III throughout the pyriform cortex and in granule cells of the caudal ventro-lateral dentate gyrus. Male Alderley Park rats, 6-8-week-old, were given a single oral dose of either 0 or 2000 mg/kg cycloate and killed for neuropathological investigation 1, 2, 3, 7, 14 or 28 days after dosing, using a regime of perfusion fixation with modified Karnovsky's fixative, followed by routine paraffin embedding. Seven transverse levels of brain were examined from each rat. Cycloate-induced neuronal cell death was seen in the pyriform cortex 1 day after dosing and persisted through to Day 28, the lesion was more marked in the rostral compared to the caudal region of the pyriform cortex. Neuronal cell death was also observed in the ventro-lateral caudal dentate gyrus on Days 1-14, day after dosing. In the early stages, Days 1-3 and to a lesser extent Day 7, the neuronal cell death resembled apoptosis, characterized by condensation of nuclear material, cell shrinkage and strong cytoplasmic eosinophilia. By Days 14 and 28 and to a lesser extent Day 7, the cell death resembled necrosis, i.e. karyorrhectic nuclei with pale irregular cytoplasm. Microglial accumulation was associated with the neuronal cell injury. In control brains, an occasional apoptotic body was seen in both the pyriform cortex and dentate gyrus. Our results demonstrate that cycloate is a novel neurotoxicant, which following a single large oral dose induces a cell specific and highly localized forebrain lesion. The time course data analyzed temporally, suggests that cycloate may cause an up regulation of apoptosis in selected regions of the adult brain.

Pathology of the liver, kidney and gonad of flounder (Platichthys flesus) from a UK estuary impacted by endocrine disrupting chemicals.

The Mersey estuary is highly contaminated with xenobiotics compared to the nearby Dee estuary. Male flounder, a migratory flatfish caught in the Mersey frequently contains high blood concentrations of the oestrogen controlled, female protein vitellogenin, suggesting that Mersey flounder have been exposed to endocrine disrupting contaminants. Males caught from the Dee contain lower blood vitellogenin levels. Preliminary histopathological examination of 410 flounder from both estuaries, focuses on the liver, kidney and gonads. Hepatic tubular vacuolation, foci of cellular alteration, and hepatocellular tumours were seen. Renal and gonadal pathology included the presence of two Mersey fish with enlarged and abnormal glomeruli, three phenotypic male Mersey flounder with unilateral intersex gonads and one male with bilateral, abnormal ovarian follicular components in the testis. These initial results provide pathological evidence of xenobiotic exposure in flounder sampled from both estuaries. Preliminary findings from flounder sampled from Millport, an offshore site in Southwest Scotland, showed no histopathological evidence of xenobiotic exposure.

Neuroprotection afforded by MK-801 against L-2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat.

Administration of a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) to rats produces marked necrosis to the granule cell layer of the cerebellum by 48 h after dosing. Associated with the neuropathology the rats show locomotor impairment and a loss of body weight and a significant increase in cerebellar water and sodium content, indicating an oedematous reaction. Cerebellar aspartate and glutamate concentrations were reduced, while glycine and glutamine concentrations were increased after this treatment. Administration of the N-methyl-D-aspartate (NMDA) receptor channel antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine (MK-801), 30 min prior to L-CPA at a dose of 0.5, 1 or 5 mg/kg i.p. prevented the necrosis to the granule cell layer of the cerebellum and the signs of motor incoordination. Similarly there was no loss in cerebellar aspartate or glutamate concentration or increase in water or sodium content. Prior treatment with MK-801 at 0.1 mg/kg did not afford protection against the neurotoxicity. Post-treatment with 1 mg/kg MK-801 up to 1 h after administering L-CPA afforded complete neuroprotection, however if delayed until 2 or 6 h it gave only partial protection, and after 12 h it gave no protection. Administration of MK-801 alone at 5 mg/kg i.p., did not alter water content, sodium concentration, aspartate or glutamate concentrations in the cerebellum. In conclusion, we have shown that MK-801 given prior to and 1 h after L-CPA can afford complete neuroprotection, suggesting that a sub-population of NMDA receptors located on granule cells in cerebellum play a key role in mediating the selective toxicity of this chemical to the rat cerebellum.

L-2-chloropropionic acid-induced neurotoxicity in the rat: a valuable model for studying selective neuronal cell death in vivo.

1. L-2-Chloropropionic acid (L-CPA) is neurotoxic when administered orally as a neutral sodium salt in high doses to rats, resulting in a selective destruction of cerebellar granule cells with the result that animals develop marked difficulties in maintaining normal locomotion. 2. Cerebellar granule cell destruction is accompanied by a reduction in cerebellar glutamate and aspartate concentrations, reductions in the density of glutamate receptors located in the cerebellar granule cell layer and development of cerebellar oedema. No other cell type in the brain, nor other organ, is affected by L-CPA. 3. The neuronal cell death is necrotic in type, involving the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors and there is some evidence for a partial role of nitric oxide in the development of the neurotoxicity. 4. Contrary to work performed on NMDA mediated cell death using neuronal cell culture approaches, L-CPA-induced granule cell death does not appear to arise from the production of excess quantities of cytotoxic free radicals, but may involve selective calcium-activated proteases, such as the calpains. 5. Tentative evidence suggests that L-CPA may interfere with voltage-dependent calcium channels in the cerebellum leading to activation of the cell death and resulting in the destruction of the granule cells. 6. In conclusion, L-CPA-induced neurotoxicity may provide valuable information on the neurochemical pathways involved in neuronal cell death that is associated with many neurological diseases.

Ultrastructural pathology and cytochemical investigations of L-2-chloropropionic acid-induced neurointoxication of the rat cerebellum.

The objectives of the studies described were to assess the ultrastructural neuropathology, blood-brain barrier (BBB) integrity and calcium status of the cerebellum of rats following a single dose of 750 mg.kg-1 L-2-chloropropionic acid (L-2-CPA). The first indications of intoxication appeared at 36 h when condensation of many granule cells associated with Purkinje cell degeneration and marked astroglial swelling were observed. Some electron-lucent granule cells were also noted lying amongst these condensed forms. Condensed granule cells had swollen, electron-lucent mitochondria, dilated Golgi apparatus and nuclear crenation. Occasionally, areas of the granule cell necrosis were also present at this time. Granule cell condensation probably represents a preliminary and irreversible stage in an excitotoxic process that leads to necrosis. At 48 and 72 h, most granule cells were necrotic, and occasionally, extravasation of both erythrocytes and leucocytes into the expanded extravascular space was observed. Evaluation of the BBB by ultrastructural cytochemical visualisation of horseradish peroxidase injected i.v. 2 min before killing by perfusion fixation showed substantial leakage. At 36 h post-dose, ultrastructural calcium localisation using oxalate/pyroantimonate precipitation demonstrated a substantial increase in calcium pyroantimonate precipitate in mitochondria and other membranous cytoplasmic organelles (especially the Golgi apparatus) in condensed granule cells, but with little in their nuclei. However, their immediate neighbours (of ostensibly normal ultrastructural appearances) contained greater amounts of intranuclear precipitate. Swollen astroglial cells (especially the Bergmann glia) contained considerable quantities of precipitate. A possible excitotoxic mechanism via L-2-CPA-induced NMDA receptor agonism leading to overwhelming calcium influx and disruption of cellular calcium homeostasis is proposed.

No changes in behaviour, nigro-striatal system neurochemistry or neuronal cell death following toxic multiple oral paraquat administration to rats.

We have examined whether the widely used herbicide, paraquat (1,1'-dimethyl-4,4'dipyridylium) may accumulate in rat brain following multiple oral dosing (5 mg paraquat ion/kg/day) for 14 days and whether this dosing regime may produce signs of neurotoxicity. This dosing regime may determine whether low dose exposure to mammals may be neurotoxic. Using [14C]paraquat to measure tissue and plasma paraquat concentrations, we observed significantly higher plasma and tissue paraquat concentrations in brain, liver, lungs and kidneys of rats which received multiple doses for 14 days, as compared to paraquat concentrations in tissues of rats which received only a single paraquat dose. Brain paraquat concentrations measured 24 h after dosing were tenfold higher in rats receiving 14 daily oral doses of paraquat, as compared to concentrations following a single oral dose. A neuropathological study of the rat brain yielded no evidence that multiple paraquat dosing resulted in neuronal cell damage. Particular attention was paid to the nigrostriatal system. The paraquat treated rats gained approximately 10% less body weight over the 15 day experimental period as compared with controls demonstrating that the dose of paraquat was toxic to the animals. Measurements of locomotor activity using open field tests or activity monitors did not reveal any statistically significant differences between control animals and those receiving paraquat. Fore- and hind-limb grip strength were not significantly different between the paraquat treated and control rats at any time point during the dosing regime, nor was there any evidence for locomotor coordination deficits in any of the animals receiving paraquat. Densities of dopamine D1 and D2, NMDA, muscarinic and benzodiazepine receptors in the cerebral cortex and striatum were not significantly different between controls and rats which had received multiple paraquat doses. Concentrations of catecholamine neurotransmitters in the striatum, hypothalamus and frontal cerebral cortex were also measured to examine whether there was evidence for catecholamine depletion in these brain regions. We did not observe any significant reductions in dopamine, noradrenaline or DOPAC concentrations in any brain region of paraquat treated rats as compared with controls. On the contrary, dopamine concentrations in the striatim were significantly elevated in paraquat treated animals following a 15 day paraquat dosing regime. We attribute these changes in catecholamine concentrations to the general toxicity of paraquat which produces a stress response. In conclusion, we could not find any evidence that multiple paraquat dosing can lead to changes in locomotor activity or grip strength. In addition, the absence of neuropathology or changes in neurochemistry in the nigrostriatal tract demonstrates that paraquat does not behave like MPP+(N-methyl-4-phenylpyridinium), the neurotoxic metabolite of MPTP.

Possible role of nitric oxide in the development of L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

1. L-2-Chloropropionic acid (L-CPA) produces selective neuronal cell necrosis in rat cerebellum when administered orally at 750 mg kg-1 that is mediated in part through activation of N-methyl-D-aspartate (NMDA) receptors. Cerebellar granule cell death occurs between 30 and 36 h following L-CPA administration exhibiting a number of features in common with excitatory amino acid-induced cell death. We have used this in vivo model to examine the neurochemical processes following L-CPA-induced activation of NMDA receptors leading to neuronal cell death in the rat cerebellum. 2. The effects of a number of compounds which potently block nitric oxide synthase in vitro were examined on L-CPA-induced neurotoxicity 48 h following L-CPA dosing, to discover whether the neuronal cell death is mediated in part by excessive nitric oxide generation. Four inhibitors were studied, NG-nitro-L-arginine (L-NOARG), NG-nitro-L-arginine methyl ester (L-NAME), NG-iminoethyl-L-ornithine (L-NIO) and 3-bromo-7-nitroindazole (BrNI). 3. L-NAME (50 mg kg-1, i.p. twice daily) and BrIN (50 mg kg-1, i.p. twice daily) administration prevented the L-CPA-induced loss of granule cells which can reach up to 80-90% of the total cell number in rats treated with L-CPA alone. L-NOARG (50 mg kg-1, i.p. twice daily) and L-NIO administered at either 25 or 100 mg kg-1, twice daily did not produce any significant protection against L-CPA-induced neurotoxicity. 4. Both L-NAME and BrIN also prevented the L-CPA-induced increase in cerebellar water content and sodium concentrations. L-NIO when administered at the highest doses prevented the increase in cerebellar sodium concentration but not water content. L-NIO and L-NOARG were ineffective in preventing the L-CPA-induced increases in cerebellar water and sodium concentrations. 5. L-CPA-induced reductions in cerebellar aspartate and glutamate concentrations and increases in glutamine and GABA concentrations were prevented by L-NAME and BrIn, but not by L-NIO or L-NOARG. Also reductions in L-[3H]-glutamate binding to glutamate ionotrophic and metabotrophic receptors in the granule cell layer of rat cerebellum was prevented by L-NAME and BrIN, but not L-NIO or L-NOARG. 6. In conclusion, the neuroprotection offered by L-NAME and BrIN suggests that L-CPA-induced cerebellar granule cell necrosis is possibly mediated by or associated with excessive generation of nitric oxide. The inability of nitric oxide synthase inhibitors, L-NOARG and L-NIO to afford protection may result from their limited penetration into the brain (L-NIO) or rapid dissociation from the enzyme.

Influence of age on the passage of paraquat through the blood-brain barrier in rats: a distribution and pathological examination.

Experiments were performed to determine the extent of paraquat entry into the brain of neonatal and elderly rats, as compared with adult rats, which may be dependent on the efficacy of the blood-brain barrier. A single, median lethal dose (20 mg/kg s.c.) of paraquat containing [14C]paraquat was administered to neonatal (10 day old), adult (3 month old) and elderly (18 month old) rats. In contrast to the adult and elderly rats where paraquat levels fell over the 24 h post-dosing period to negligible levels, paraquat concentrations in neonatal brains did not decrease with time between 0.5 and 24 h following dosing. The distribution of [14C]paraquat was measured in selective brain regions using quantitative autoradiography in all three age groups of rats, 30 min and 24 h following dosing. Autoradiography demonstrated that brain paraquat distributions were similar in the rat age groups. Most of the paraquat was confined to regions outside the blood-brain barrier and to brain regions that lack a complete blood-brain barrier e.g. dorsal hypothalamus, area postrema and the anterior olfactory bulb. Between 0.5 h and 24 h following dosing, paraquat concentrations in deeper brain structures, some distance away from the sites of entry, began to slowly increase in all the rat age groups. By 24 h following dosing, a majority of brain regions examined using quantitative autoradiography revealed significantly higher paraquat concentrations in neonatal brains as compared to brain regions of adult and elderly rats. Despite increased paraquat entry into neonatal brain, we could find no evidence for paraquat-induced neuronal cell damage following a detailed histopathological examination of perfused-fixed brains. In conclusion, impaired blood-brain barrier integrity in neonatal brain thus permitting more paraquat to enter than in adult brain, did not result in neuronal damage.

Neuropathological changes in rat brain following oral administration of 2-chloropropionic acid.

The agrochemical intermediate, L-2-Chloropropionic acid (L-2-CPA) and D-2-chloropropionic acid (D-CPA), when administered separately by oral gavage to rats, produced extensive cerebellar granule cell necrosis (> 80%) characterised by varying degrees of nuclear condensation and nuclear karyorrhexis. In contrast a few necrotic Purkinje cells (< 5%) were observed. Purkinje cell damage consisted of cytoplasmic and nuclear shrinkage and hyperchromasia. Karyorrhexis was not seen in Purkinje cells. Extensive vacuolation (edema) was present both in the cerebellar granular layer and Purkinje cell layer. Astrogliosis (hypertrophy and hyperplasia) was seen at lesion sites and activity was recognised by positive glial fibrillary acidic protein (GFAP) staining. Proliferative activity of astrocytes at lesion sites was confirmed by positive proliferating cell nuclear antigen (PCNA) immunostaining. Astrogliosis was focused exclusively in the necrotic granule cell layer. A single oral dose of 750 mg/kg of either stereoisomer or three consecutive daily doses of 250mg/kg L-2-CPA. produced the lesion. Cerebellar water content increased with time in parallel with the edematous response noted by neuropathological examination. The earliest onset of the lesion was observed at 36 hours appearing more extensive at 48 and 72 hours post-dosing. No other major neuropathological changes were detected in the brain, spinal cord, spinal ganglia, Gasserian ganglia, peripheral nerves and voluntary muscle following L-2-CPA administration. In conclusion, both stereoisomers of CPA were found to be selective neurotoxicants of the rat central nervous system.

The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat.

The role of glutathione (GSH) in the neurotoxicity produced following a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) has been investigated in rats. L-CPA-induced neurotoxicity was characterised by up to 80-90% loss in cerebellar granule cells and cerebellar oedema leading to locomotor dysfunction. Neurochemically, L-CPA-induced neurotoxicity produced a reduction in the concentration of aspartate and glutamate in the cerebellum and a reduction in the density of NMDA receptors in the cerebellar cortex, whilst there was an increase in cerebellar glycine, glutamine and GABA concentrations. Treatment of rats with buthionine sulfoximine (BSO) at 1 g/kg, i.p., an inhibitor of GSH synthesis, potentiated the toxicity of L-CPA, such that many of the neurochemical markers were significantly different from controls at earlier time points, compared to animals which had received L-CPA alone, and toxicity was also seen in the kidney of BSO plus L-CPA treated rats. In contrast, supplementing GSH concentrations by administration of the isopropyl ester of glutathione (i.p.-GSH) at 1 g/kg, s.c., was able to protect rats against L-CPA neurotoxicity and prevent many of the neurochemical changes. In order to assess whether the depletion of GSH in the rat cerebellum following L-CPA treatment was related to the delivery of cysteine or cystine, the accumulation of [14C] cystine into cerebellar slices was characterised and found to be energy dependent, Na+ independent and obey saturation kinetics with an apparent Km of 77 microM and an apparent Vmax of 450 nmol/g wet weight per h. The accumulation of cystine into cerebellar slices was non-competitively inhibited by the cysteine conjugate of L-CPA with an apparent K(i) of approximately 60 microM, whilst glutamate only inhibited cystine accumulation at doses which were cytotoxic to cerebellar slices. Hence the depletion of GSH in the rat cerebellum, following L-CPA administration, may be due to a reduction in the delivery to the brain of cysteine or cystine, one of the components required for GSH synthesis, by the cysteine conjugate of L-CPA. Our studies show the pivotal role GSH plays in cerebellar granule cell necrosis induced by L-CPA in the rat, indicating that a marked and sustained reduction in cerebellar GSH content by L-CPA may leave granule cells vulnerable to cytotoxic free radical damage leading to cell death, possibly mediated through excitatory amino acids.

L-2-chloropropionic acid-induced neurotoxicity is prevented by MK-801: possible role of NMDA receptors in the neuropathology.

We have demonstrated that following a single oral dose of L-2-chloropropionic acid (L-CPA) to rats (750 mg/kg; pH 7) there was a marked and widespread loss of granule cells in the cerebellum as assessed by neuropathology by 48 hr. There also appeared to be limited damage to Purkinje cells, whereas stellate, Golgi, and basket cells were not affected by L-CPA administration. The L-CPA-mediated cerebellar neuropathology was accompanied by a significant increase in the cerebellar water content and sodium concentration, 48 hr following L-CPA administration, suggesting an edematous reaction. After 36 hr, the animals displayed marked locomotor dysfunction and had to be terminated at 54 hr due to marked weight loss. We did not observe any neuropathology in forebrain regions nor was the water content in the forebrain significantly different from controls in animals which had been treated with L-CPA. Cerebellar aspartate concentrations were reduced 48 hr following L-CPA administration becoming marked at 54 hr and accompanied by a significant reduction in cerebellar glutamate concentrations. The density of N-methyl-D-aspartate (NMDA) receptors in the granular layer of the cerebellar cortex was also significantly reduced at 48 and 54 hr following L-CPA administration. Prior administration of MK-801 (dizocilpine) (5 mg/kg/i.p.), an irreversible NMDA receptor antagonist, 30 min before an oral dose of L-CPA (750 mg/kg) prevented the loss of both granule and Purkinje cells. There was no abnormal locomotor activity in the L-CPA rats treated with MK-801 except for the first 4 hr following dosing when animals were severely sedated. Animals which received L-CPA plus MK-801 were normal 96 hr post dosing showing that MK-801 did not delay the onset of L-CPA toxicity. There was no alteration in cerebellar water content or sodium concentrations in rats which had been administered MK-801 with L-CPA. The reductions in cerebellar aspartate and glutamate concentrations were totally prevented by administration of MK-801, as was the reduction in L(-)[3H]glutamate binding to cerebellar NMDA receptors. Administration of MK-801 alone (5 mg/kg/i.p.) did not alter the water content, sodium concentrations, aspartate or glutamate concentrations, or the density of NMDA receptors in the cerebellum. In conclusion, we suggest that L-CPA-induced neurotoxicity leading to loss in granule cells and an accompanying cerebellar edema can be prevented by MK-801, suggesting that a subpopulation of NMDA receptors found on cerebellar granule cells play a pivotal role in mediating the toxicity of this compound.

Changes in cerebellar amino acid neurotransmitter concentrations and receptors following administration of the neurotoxin L-2-chloropropionic acid.

1p4studied the effect of L-2-chloropropionic acid (L-CPA)-induced (250 mg/kg/po/day for 3 days) neurotoxicity, which results in an almost total destruction of cerebellar granule cells over 5 days, on forebrain and cerebellar neurochemistry. There was a reduction in cerebellar aspartate and glutamate concentrations of L-CPA-treated rats and a reduction in N-methyl-D-aspartate (NMDA) and kainate receptor densities in the cerebellar cortex following loss of the granule cells. Concentrations of glutamine and GABA were increased transiently during the development of the granule cell lesion but fell back to control levels by Day 5 of the study. Glycine concentrations began to rise as the granule cells began to disappear and concentrations remained elevated until the end of the study. In contrast, concentrations of taurine began to fall around the same time point as the granule cells were gradually depleted. We did not observe any consistent changes in forebrain amino acid concentrations following the L-CPA administration or any changes in NMDA, kainate, GABAA, or A1-adenosine receptor densities. We therefore conclude that the L-CPA-induced loss in cerebellar granule cells is accompanied by a reduction in cerebellar aspartate and glutamate concentrations and in the density of NMDA and kainate receptors in the cerebellar cortex. Changes in cerebellar GABA, glutamine, glycine, and taurine concentrations probably reflect secondary compensatory changes in cerebellar activity resulting from a widespread loss of cerebellar granule cells and loss of excitatory inputs. We suggest that L-CPA-induced neurotoxicity may be valuable tool to study cerebellar neurochemistry and physiology.

Further evidence that the blood/brain barrier impedes paraquat entry into the brain.

The distribution of the non-selective herbicide paraquat was examined in the brain following subcutaneous administration of 20 mg kg-1 paraquat ion containing [14C]paraquat to male adult rats in order to determine whether paraquat crosses the blood/brain barrier. Following administration, [14C]paraquat reached a maximal concentration in the brain (0.05% of administered dose) within the first hour and then rapidly disappeared from the brain. However, 24 h after administration of the herbicide, about 13% of the maximal recorded concentration of paraquat remained in the brain (1.6 nmol g-1 wet weight) and could not be removed by intracardiac perfusion. Using measurements of [14C]paraquat in dissected brain regions and using quantitative autoradiography we demonstrated an asymmetrical distribution in and around the brain at 30 min (maximal concentration) and 24 h after administration. Most of the paraquat was associated with five structures, two of which, the pineal gland and linings of the cerebral ventricles lie outside the blood/brain barrier whilst the remaining three brain areas, the anterior portion of the olfactory bulb, hypothalamus and area postrema do not have a blood/brain barrier. Overall, the distribution of [14C]paraquat in the brain 24 h after systemic administration was highly correlated to the blood volume. These data indicate that any remaining paraquat in the brain 24 h after systemic administration is associated with elements of the cerebro-circulatory system, such as the endothelial cells that make up the capillary network and that there is a limited entry of paraquat into brain regions without a blood/brain barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

[125I]endothelin binding in rat cerebellum is increased following L-2-chloropropionic-acid-induced granule cell necrosis.

The systemic administration of L-2-chloropropionic acid (L-CPA) to rats produced a marked depletion of cerebellar granule cells (> 80% of the total) when administered in a single oral dose of 750 or 250 mg/kg/day for 3 days. The nature of the L-CPA-induced neurotoxicity is currently unknown but it exhibits a number of features in common with excitatory amino acid-induced neuronal cell death. We observed an increase in [125I]endothelin-1 (ET-1) binding in the cerebellar cortex, as measured by quantitative receptor autoradiography, which occurs at 48 h, but not 24 h, following the 750 mg/kg L-CPA dosing regimen. The increase in [125I]ET-1 binding did not parallel the cellular damage and resultant astrocyte proliferation, as measured by GFAP immunoreactivity, which was primarily confined to the granular layer of the cerebellar cortex. The increased [125I]ET-1 binding occurred in the molecular layer of the cerebellar cortex (controls 2.03 +/- 0.26 fmol/mg tissue; L-CPA-treated 6.69 +/- 0.45 fmol/mg tissue, n = 6; p < 0.01, Student's t-test) which appeared to contain astrocytic processes originating from the large increase in astrocyte number situated in the granular layer. Pretreatment of the rats with the irreversible NMDA receptor antagonist, MK-801, protected the cerebellar granule cells against the L-CPA neurotoxicity and also prevented the increase in [125I]ET-1 binding in the cerebellar cortex. The increased [125I]ET-1 binding in rat cerebellum appears to be linked to the reactive gliosis that occurs in association with neuronal cell injury.

Induction of trimethyltin neurotoxicity by dietary administration.

Groups of 12 male and 12 female rats were fed diets containing 0 or 8ppm trimethyltin chloride for up to 25 days. All the animals were observed prior to the study start and daily throughout the study for any changes in clinical condition. In addition, detailed clinical observations, including quantitative assessments of landing foot splay, sensory perception, muscle weakness and locomotor activity were monitored during the study. At the end of the study, the rats were killed and subjected to a full Post-mortem. Selected nervous system tissues were removed, processed and examined microscopically. Clinical signs typical of trimethyltin neurotoxicity (e.g. aggression, shaking and convulsions) were seen in rats receiving diets containing 8ppm trimethyltin chloride for as little as 22 days. Neuropathological lesions consisting of extensive neuronal cell necrosis in the limbic region of the brain, vacuolar degeneration of ventral horn cells of the spinal cord and a marginal increase in Wallerian-type degeneration were seen. The study demonstrates that trimethyltin neurotoxicity can be induced by dietary administration and that both male and female rats are equally sensitive.

Upregulation of the POMC gene in rats by a neurotoxicant which targets motoneurones.

Immunocytochemistry and histochemical in situ hybridisation were used to detect POMC-derived peptides (beta-endorphin and alpha-MSH) and POMC mRNA respectively, in the lumbar spinal cord of adult rats. In normal rats the incidence of ventral horn cells which expressed the peptides or mRNA was negligible. However after treatment with IDPN a neurotoxicant which targets the motoneurones the peptides and POMC mRNA were detectable in over 65% of ventral horn motoneurones. After treatment with 1,3 DNB a neurotoxicant which does not target motoneurones immunostaining for the peptides and mRNA was negligible. Thus chemical intoxication of the motoneurones causes upregulation of the POMC gene.

Effect of IDPN on the expression of POMC-derived peptides in rat motoneurones.

Immunocytochemistry was used to detect beta-endorphin and alpha-melanotropin (alpha-MSH) in lumbar spinal motoneurones in rats treated with beta,beta'-iminodiproprionitrile (IDPN), a neurotoxicant that targets motoneurones or corn oil, which has no known neurotoxicity. After IDPN treatment most of the motoneurones were immunoreactive for both peptides but after corn oil treatment immunostaining was negligible. It is suggested that increased expression of the POMC-derived peptides may be part of the regenerative repertoire of the damaged motoneurone regardless of the cause of the lesion. Alternatively the peptides may simply accumulate in the motoneurones as a result of impaired axoplasmic transport.

Mechanism of action of triethyltin on identified leech neurons.

The effects of triethyltin (TET) have been examined using intracellular electrophysiological recording techniques from identified neurons of the leech (Hirudo medicinalis) CNS and from salivary glands of the giant Amazon leech (Haementeria ghilianii). TET, at concentrations as low as 10(-5) M, caused a reversible neuronal membrane depolarisation accompanied by an increase in firing frequency of action potentials (which could lead to conduction block at 10(-4) M) and a concomitant decrease in membrane resistance. TET-induced membrane depolarisation still occurred in saline where Na+, K+ and Ca2+ had been replaced by choline. TET decreased the rate of the depolarising and repolarising phases of the action potential. This also occurred in Haementeria salivary gland cells, in which the only inward cation channel is a calcium channel. The calcium channel blocker, manganese, did not block the effects of TET. TET counteracted the effects on the action potential of the potassium channel blocker, tetraethylammonium chloride (TEA). TET-induced neurotoxicity occurred independently of any resultant toxic effects on the myelin sheath. The action of TET is consistent with our view that it causes an increase of intracellular free Ca2+ probably via release from intracellular stores and inhibition of Ca2+ reuptake. A resulting inhibition of the Na+/K+ and Ca2+ pumps may also occur.

Alpha 2U-globulin: measurement in rat kidney and relationship to hyaline droplets.

The concentration of renal alpha 2U-globulin increased in a dose-dependent manner in adult male but not female rats which received a single dose of 2,2,4-trimethylpentane (TMP). After administration of a single dose of 12 mmol TMP/kg to adult male rats, the renal concentration of alpha 2U-globulin reached a peak at 48 hours and returned to near background level after 7 days. These changes in renal alpha 2U-globulin concentration were closely paralleled by changes in renal hyaline droplet formation. Renal alpha 2U-globulin and hyaline droplets were absent in normal pre-puberty male rats, and neither could be stimulated by a single dose of TMP. alpha 2U-Globulin was localised in the renal cortex of adult male rats, in particular the S2 segment of the proximal tubule. A greater staining intensity due to alpha 2U-globulin was seen in the S2 and adjacent segments after a single dose of TMP. A strong association is suggested between the presence of renal hyaline droplets and the occurrence of alpha 2U-globulin.

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane.

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.