Reduced expression of the KATP channel subunit, Kir6.2, is associated with decreased expression of neuropeptide Y and agouti-related protein in the hypothalami of Zucker diabetic fatty rats.

The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.

Continuous inhibition of 11ß-hydroxysteroid dehydrogenase type I in adipose tissue leads to tachyphylaxis in humans and rats but not in mice.

BACKGROUND AND PURPOSE: 11ß-hydroxysteroid dehydrogenase type I (11ß-HSD1), a target for Type 2 diabetes mellitus, converts inactive glucocorticoids into bioactive forms, increasing tissue concentrations. We have compared the pharmacokinetic-pharmacodynamic (PK/PD) relationship of target inhibition after acute and repeat administration of inhibitors of 11ß-HSD1 activity in human, rat and mouse adipose tissue (AT). EXPERIMENTAL APPROACH: Studies included abdominally obese human volunteers, rats and mice. Two specific 11ß-HSD1 inhibitors (AZD8329 and COMPOUND-20) were administered as single oral doses or repeat daily doses for 7-9 days. 11ß-HSD1 activity in AT was measured ex vivo by conversion of (3) H-cortisone to (3) H-cortisol. KEY RESULTS: In human and rat AT, inhibition of 11ß-HSD1 activity was lost after repeat dosing of AZD8329, compared with acute administration. Similarly, in rat AT, there was loss of inhibition of 11ß-HSD1 activity after repeat dosing with COMPOUND-20 with continuous drug cover, but effects were substantially reduced if a 'drug holiday' period was maintained daily. Inhibition of 11ß-HSD1 activity was not lost in mouse AT after continuous cover with COMPOUND-20 for 7 days. CONCLUSIONS AND IMPLICATIONS: Human and rat AT, but not mouse AT, exhibited tachyphylaxis for inhibition of 11ß-HSD1 activity after repeat dosing. Translation of observed efficacy in murine disease models to human for 11ß-HSD1 inhibitors may be misleading. Investigators of the effects of 11ß-HSD1 inhibitors should confirm that desired levels of enzyme inhibition in AT can be maintained over time after repeat dosing and not rely on results following a single dose.

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.

Failure of glycine site NMDA receptor antagonists to protect against L-2-chloropropionic acid-induced neurotoxicity highlights the uniqueness of cerebellar NMDA receptors.

Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of excitatory amino acid-induced cell death mediated in part by the activation of NMDA receptors. However, the NMDA subunit stoichiometry, producing functional NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the NMDA receptors in forebrain regions. We have used the L-2-chloropropionic acid (L-CPA) (750 mg/kg) model of NMDA-mediated selective cerebellar granule cell necrosis in vivo to examine the role of the glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell necrosis. The abilities of various NMDA receptor antagonists were examined in vivo to determine the relative contribution of both glutamate and glycine sites involved in the L-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive NMDA receptor antagonist dizocilpine (MK-801) was compared with glutamate and glycine site NMDA antagonists. We have examined a number of markers for the L-CPA-induced granule cell necrosis. The L-CPA-induced reduction in cerebellar aspartate and glutamate concentrations were used as markers of granule cell necrosis. We also measured the cerebellar water content and sodium concentrations as measures of the L-CPA-induced cerebellar edema that accompanies the granule cell necrosis. Finally the ability of the NMDA antagonists to attenuate the L-CPA-induced reductions in body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent glutamate antagonists, CPP and CGP40116 and dizocilpine prevented the L-CPA-induced locomotor dysfunction and granule cell necrosis as measured by their ability to prevent L-CPA-induced reduction in aspartate and glutamate concentrations. CPP, CGP40116 and dizocilpine also prevented the appearance of cerebellar edema following L-CPA administration. In addition, dizocilpine, CPP and CGP40116 were able to partially prevent the L-CPA-induced loss in body weight over the 48 h experimental period. In contrast, none of the glycine partial agonists or antagonists, namely (+/-)HA-966, D-cycloserine, MDL-29,951, DPCQ, MNQX or L-701 252 were able to prevent the L-CPA-induced loss in body weight, L-CPA-induced granule cell necrosis and behavioral disturbances when administered to rats. None of the NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for dizocilpine, CPP, CGP40116 and (+/-)HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrated that NMDA open channel blockade and glutamate antagonists can provide full neuroprotection against the L-CPA-induced granule cell necrosis. The failure of the glycine partial agonist and antagonists to provide any neuroprotection against L-CPA-induced neurotoxicity in the cerebellum contrast with their neuroprotective efficacy in other animal models of excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the glycine site plays a lesser role in modulating NMDA receptor function in the cerebellum and may explain why cells expressing NMDA receptors composed of NR1/NR2C subunits are particularly resistant to excitatory amino acid-induced neurotoxicity.

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.

2-Halopropionic acid-induced cerebellar granule cell necrosis in the rat: in vivo and in vitro studies.

Daily oral administration of 2.3 mmol/kg L-2-chloropropionic acid (L-2-CPA), DL-2-bromopropionic acid (2-BPA) or DL-2-iodopropionic acid (2-/PA) but not DL-2-fluoropropionic acid (2-FPA) produced cerebellar granule cell necrosis in the rat. Twenty four hours after three doses of L-2-CPA or two doses of 2-BPA, animals showed clinical signs of motor incoordination and reduced hindlimb function which was associated with marked cerebellar oedema and cerebellar granule cell necrosis. Biochemical analyses showed a marked increase in cerebellar water and Na+ content, and a reduction in cerebellar glutamate and aspartate. 2-IPA at this dose was toxic, the animals not surviving a second dose, histopathology showed hepatic and renal necrosis with mild cerebellar granule cell necrosis. 2-FPA was not neurotoxic after four daily doses. A marked decrease in hepatic and cerebellar non-protein sulphydryl (NP-SH) content was observed 4 h after a single dose of 2.3 mmol/kg L-2-CPA, 2-BPA and 2-IPA but not 2-FPA. Daily doses of 2-BPA for 3 days produced a sustained 50% depletion in cerebellar NP-SH. In vitro, L-2-CPA, 2-BPA and 2-IPA produced glutathione (GSH) depletion in the presence of rat liver cytosol, while 2-FPA did not. Depletion of GSH in the presence of cerebellar cytosol was only observed with 2-IPA. Studies using primary cultures of rat cerebellar granule cells, showed that all analogues produced a concentration dependent loss of cell viability. Mean EC50 values for 2-FPA, L-2-CPA, 2-BPA and 2-IPA toxicity were 1.7, >10, 0.5 and 0.3 mM, respectively, for 24 h continuous exposure. MK-801 and Vitamin E afforded protection against L-2-CPA-induced cytotoxicity but not against the other analogues. In summary, in addition to L-2-CPA, both 2-BPA and 2-IPA produce cerebellar granule cell necrosis in the rat. Depletion of GSH in the cerebellum may be contributory factor in the cascade of events leading to neurotoxicity.

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 absence of cerebellar granule cell necrosis in the mouse following L-2-chloropropionic acid administration.

Oral administration of L-2-chloropropionic acid (L-CPA) to rats either as a single dose (750 mg/kg) or daily doses (250 mg/kg per day for 3 days) produces selective necrosis to the granule cell layer of the cerebellum. As part of a study to understand the mechanism of this selective toxicity, we investigated the toxicity of L-CPA and a related analogue, DL-2-bromopropionic acid to the mouse with particular emphasis on the brain. Following a single oral dose (up to 1000 mg/kg), or daily oral doses of 250 mg/kg per day L-CPA up to maximum tolerated doses, produced no evidence of neurotoxicity. Similarly, daily oral doses of DL-2-bromopropionic acid at 250 mg/kg per day produced no evidence of neurotoxicity. The basis for the lack of response was explored by examining the metabolism and disposition of L-[2-14C]-CPA in the mouse. Following a single oral dose of 250 mg/kg L-CPA, radioactivity was rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of radiolabel and L-CPA occurred within 2 h of dosing at about 1.8 mM, and were then lost from the plasma with a half-life of 1 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid derived from the glutathione conjugate. About 39% of the dose was excreted in the urine in the first 24 h, mainly as 2-S-cysteinylpropanoic acid with only a small amount of unchanged L-CPA. The remaining radiolabel from L-CPA was excreted in the faeces (26%) and exhaled as carbon dioxide (about 14%) over 72 h. Radiolabel from L-[2-14C]-CPA was present in the cerebellum at a peak concentration of 1 mM 1-2 h after dosing and then was lost more slowly than from the plasma. Measurement of non-protein sulphydryl content in the brain, liver and kidneys showed a decrease in the liver and kidneys 4 h after dosing which recovered fairly rapidly, while a more prolonged decrease was found in the brain, especially the cerebellum. Our studies show that the mouse is refractory to cerebellar injury following treatment with L-CPA and DL-2-bromopropionic acid. The mouse appears to metabolize and excrete L-CPA as its glutathione-derived conjugate(s) more rapidly than the rat, thereby limiting the availability of L-CPA to the cerebellum, which may account for the absence of neuronal cell injury.

The effect of postnatal age on L-2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat.

L-2-Chloropropionic acid (L-2-CPA) selectively damages the cerebellum in adult rats. The rat cerebellum continues to develop postnatally during the first 4 weeks of life. In this study we examined the neurotoxic effect on rats of increasing postnatal age. Daily oral dosing of rats aged 56 days with 250 mg/kg per day of L-2-CPA for 3 days produced necrosis to neurons in the cerebellar granule cell layer and to neurons in the medial/ventral region of the habenular nucleus. Rats aged 22 days were resistant to the cerebellar toxicity while rats aged 32 days and older were sensitive. A single large oral dose of 500 or 750 mg/kg L-2-CPA produced no clinical signs of neurotoxicity or lesions in the cerebellum 48 h after dosing in 22-day-old rats. Daily dosing of 22-day-old rats at 250 mg/kg per day L-2-CPA for 10 days also produced no signs of neurotoxicity or reduction in body weight gain, although histological examination of the brain revealed slight neuronal cell necrosis in the granule cell layer of the cerebellum with a minimal effect in the medial/ventral region of the habenular nucleus. In contrast, daily dosing of rats aged 32, 38, 48 and 58 days with 250 mg/kg per day of L-2-CPA for 3 days produced clear signs of neurotoxicity which were associated with reduced body weight gain and loss of hindlimb function. In these rats there was clear evidence of neuronal cell loss in the cerebellar granule cell layer and medial/ventral region of the habenular nucleus. This study showed that the postnatal developing cerebellum is resistant to L-CPA-induced injury in rats up to 25 days of age, but becomes vulnerable to the toxicity by 32 days of age. The basis for the resistance of the developing cerebellum to L-CPA is discussed.

L-2-Chloropropionic acid metabolism and disposition in male rats: relevance to cerebellar injury.

L-2-Chloropropionic acid (L-CPA) produces selective necrosis to the granule cell layer of the rat cerebellum. As part of a study to understand the mechanism of selective toxicity we have investigated the metabolism and disposition of [2-14C]L-CPA in the rat, with particular emphasis on the brain. Following a single oral non-toxic dose of 250 mg/kg or a neurotoxic dose of 750 mg/kg or 250 mg/kg per day for 3 days, L-CPA is very rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of 2 mM (250 mg/kg) and 6 mM (750 mg/kg) L-CPA occurred within 1 h of dosing, and the compound was readily cleared from the plasma with a half-life of 2.6 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid, presumably derived from the glutathione conjugate. About 60% of the dose is excreted in the urine in the first 24 h as unchanged L-CPA, with a smaller amount excreted as the mercapturate, 2-S-N-acetylcysteinylpropanoic acid. Little radiolabel from L-CPA is excreted in the faeces; however, approximately 18% of a 250 mg/kg dose of L-CPA is eliminated as carbon dioxide. The radiolabel from [2-14C]L-CPA present in the cerebellum, forebrain and liver at all time intervals examined was L-CPA. There was some indication of retention of L-CPA in the brain relative to the plasma with a small but consistently higher concentration found in the cerebellum. Whole body autoradiography studies indicated some selective retention of radiolabel in the cerebellum after the third dose of 250 mg/kg [2-14C]L-CPA. Our findings indicate that the initial insult to the cerebellum following L-CPA administration is probably due to the parent compound however, the prolonged presence of 2-S-cysteinylpropanoic acid in the plasma and concomitant depletion of glutathione in the cerebellum may also play a role in the toxicity. The relevance of the slightly greater retention of L-CPA in the cerebellum to the selective neurotoxicity of L-CPA requires further study.

Glutathione depletion in the liver and brain produced by 2-chloropropionic acid: relevance to cerebellar granule cell necrosis.

L- and D-2-chloropropionic acid (L-CPA and D-CPA) produce selective damage to granule cells of the rat cerebellum by a mechanism that is not currently understood. We have demonstrated that both L- and D-CPA produce a rapid, dose and time dependent depletion of liver non-protein sulphydryl (NP-SH) content, mainly glutathione (GSH), while in the cerebellum and forebrain, there is a slower, dose and time dependent decrease in NP-SH. Twenty-four hours after a single dose of 750 mg/kg L-CPA (a dose sufficient to produce cerebellar toxicity, but a time prior to the onset of cellular necrosis), the content of total GSH was depleted by 85% in the cerebellum and to a lesser degree in the forebrain, while no increase in oxidised glutathione was observed in either tissue. In vitro both L- and D-CPA caused a marked reduction in GSH concentration when incubated with hepatic cytosol but not hepatic microsomes or brain cytosol. The hepatic cytosolic depletion appeared to be due to a direct reaction catalysed by a theta class glutathione S-transferase. A GSH adduct of L-CPA was isolated by high pressure liquid chromatography and identified by mass spectrometry as 2-S-glutathionyl propanoic acid, confirming a direct substitution reaction. No GSH adducts were formed by cerebellar or forebrain cytosol, suggesting that the particular isoform of glutathione S-transferase catalysing the reaction may not be present in the brain. We suggest that the marked and sustained CPA-mediated GSH depletion in the granule cells of the cerebellum may render these cells more vulnerable to oxidative free radical damage.

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.

Calpain activation and not oxidative damage mediates L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

Possible biochemical events involved in L-2-chloropropionic acid (L-CPA)-induced delayed cerebellar granule cell necrosis following N-methyl-D-aspartate activation were studied in vivo. We examined whether the calcium-sensitive proteolytic enzymes, the calpains, may be activated by L-CPA or whether the generation of excess quantities of cytotoxic free radicals may play a role in the neurotoxicity produced by oral administration of L-CPA (750 mg/kg, pH 7.0). Evidence for free radical-induced cellular damage was examined using biochemical approaches such as examining brains from L-CPA-treated rats for increased lipid peroxidation, DNA damage, or protein oxidation. Second, the ability of antioxidants to provide neuroprotective activity against L-CPA-induced neurotoxicity was examined in vivo. Western blotting using antibodies against spectrin (alpha-fodrin) demonstrated evidence for calpain (EC 3.4.22.17) activation in the cerebellum, but not in the cerebral cortex of L-CPA-treated rats at 36 and 48 hr after L-CPA dosing. In contrast, there was no evidence for oxidative damage to cerebellar proteins or lipids in L-CPA-treated rat brains compared to controls. We also could not find evidence for DNA damage using the TUNEL method for the detection of single- and/ or double-strand breakage in situ in L-CPA-treated brains. We examined whether a number of reported antioxidants may be effective against L-CPA-induced neurotoxicity. The aminosteroids U74389G and U83836E, the free radical scavengers 3-methyl-1-phenylpyrazolin-5-one and N-tert-butylphenylnitrone, and the iron chelator N-ethoxy-2-ethyl-3-hydroxypyridin-4-one were all ineffective in attenuating L-CPA neurotoxicity. We suggest that L-CPA-induced cerebellar necrosis is the result of calpain activation which results in the degradation of cytoskeletal proteins and other proteins necessary for cellular biochemistry. We could find no evidence of oxidative damage to cerebellar proteins, lipids, or DNA as a result of excess amounts of free radicals, and selective antioxidants were unable to provide neuroprotection against L-CPA neurotoxicity, suggesting that oxidative stress does not play a role in the granule cell necrosis.

Chloropropionic acid-induced alterations in glucose metabolic status: possible relevance to cerebellar granule cell necrosis.

We have examined the effect of L- and D-2-chloropropionic acid (L-CPA and D-CPA) on the concentrations of pyruvate, lactate, glucose and beta-hydroxybutyrate in the blood at various times after doses which produce cerebellar granule cell necrosis. Blood pyruvate and lactate concentrations were reduced in these animals 4 h after dosing and remained below those of controls for up to 48 h. No changes were seen in concentrations of plasma glucose of beta-hydroxybutyrate at any time point. Similarly, no changes in cerebellar glucose, pyruvate or lactate were seen 24 h after 750 mg/kg L-CPA. Oxidation of [14C] pyruvate or [14C] glucose to [14CO2] by cerebellar slices from control rats was not altered by the presence of L- or D-CPA (1-10 mM for 2 h). Nor were these parameters affected in cerebellar slices from rats killed 24 h after a dose of 750 mg/kg L-CPA. We conclude that the activation of the pyruvate dehydrogenase complex is unlikely to be a critical factor in the selective toxicity of CPA to the cerebellum.

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.

L-2-chloropropionic acid-induced cerebellar granule cell necrosis is potentiated by L-type calcium channel antagonists.

We have used the model of L-2-chloropropionic acid (L-CPA)-induced selective cerebellar granule necrosis to study excitatory amino acid-induced necrotic cell death in vivo produced by the activation of N-methyl-D-aspartate (NMDA) receptors. However, the mechanism for the NMDA receptor activation and the biochemical events which dictate the anatomical selectivity for the L-CPA-induced lesion are as yet unknown. We examined whether blockade of sodium and calcium channels may reduce the neurotoxicity through a reduction of glutamate release from granule cells. None of the sodium channel antagonists examined, i.e. phenytoin, lamotrigine or rilazole nor the mixed sodium/calcium channel blocker, lifarazine, altered the L-CPA neurotoxicity. However, L-type calcium channel blockers, verapamil and nifedipine enhanced the L-CPA-induced granule cell necrosis, assessed by measuring the degree of L-CPA-induced reductions in cerebellar aspartate concentration, increases in cerebellar glycine concentrations and the development of cerebellar oedema. In addition, the locomotor activity of rats receiving both L-CPA and either verapamil or nifedipine was significantly lower than when rats received L-CPA alone, suggesting an enhancement of the neurotoxicity of L-CPA by L-type calcium channel blockade. The data suggest that L-CPA may interfere with non-L-type calcium channels located on granule cell bodies and nerve terminals leading to reduction of the calcium entry into the cells. We suggest that a combination of L-type channel blockade and non-L-type channels which are sensitive to L-CPA produces reductions in intracellular calcium concentrations below that required for neuronal survival.