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E A Lock

Publications and source records attributed to E A Lock.

At least 37 records · Page 2Linked to original sources
PubMed1999

Symposium overview: the role of glutathione in neuroprotection and neurotoxicity.

Although the cytoprotective effects of glutathione (GSH) are well established, additional roles for GSH in brain function are being identified that provide a pharmacological basis for the relationship between alterations in GSH homeostasis and the development of certain neurodegenerative processes. Thus, GSH and glutathione disulfide (GSSG) appear to play important functional roles in the central nervous system (CNS). A symposium, focussing on the emerging science of the roles of GSH in the brain, was held at the 37th annual meeting of the Society of Toxicology, with the emphasis on the role of glutathione in neuroprotection and neurotoxicity. Jean Francois Ghersi-Egea opened the symposium by describing the advances in our understanding of the role of the blood-brain and blood-cerebral spinal fluid (CSF) barriers in either limiting or facilitating the access of xenobiotics into the brain. Once within the brain, a multitude of factors will determine whether a chemical causes toxicity and at which sites such toxicity will occur. In this respect, it is becoming increasingly clear that GSH and its various conjugation enzymes are not evenly distributed throughout the brain. Martin Philbert discussed how this regional heterogeneity might provide a potential basis for the theory of differential sensitivity to neurotoxicants, in various regions of the brain. For certain chemicals, GSH provides neuroprotection, and Edward Lock discussed the selective toxicity of 2-chloropropionic acid (CPA) to the cerebellum and how its modification by modulating brain thiol status provides an example of GSH acting in neuroprotection. The sensitivity of the cerebellum to CPA may also be linked to the ability of this compound to activate a sub-type of the NMDA receptor. Thus, GSH and cysteine alone, or perhaps as conjugates with xenobiotics, may play a role in excitotoxicity via NMDA receptor activation. In contrast, certain chemicals may be converted to neurotoxicants following conjugation with GSH, and Arthur Cooper described how the pyridoxal 5'-phosphate-dependent, cysteine conjugate beta-lyases might predispose the brain to chemical injury in a GSH-dependent manner. The theme of GSH as a potential mediator of chemical-induced neurotoxicity was extended by Terrence Monks, who presented evidence for a role for GSH conjugation in (+/-)-3,4- methylenedioxyamphetamine-mediated serotonergic neurotoxicity.

Animals↗
PubMed1998

Investigations of the pathways of toxicity of methyl iodide in the rat nasal cavity.

The monohalomethane methyl iodide (MeI) is a site specific toxin within the nasal cavity of the rat, selectively damaging the olfactory epithelium (OE) whilst respiratory epithelium (RE) is spared. The aim of this study was to investigate the rates and routes of metabolism of MeI within the nasal cavity, in order to understand the reasons for the observed site-selectivity. Cytosolic glutathione S-transferases (GSTs) of both OE and RE catalysed the conjugation of MeI with glutathione (GSH), but rates were 4-fold higher in OE than RE. The product of this reaction was confirmed as S-methyl GSH. In both OE and liver the GST catalysing the conjugation of MeI was shown to belong to the theta class. No cytochrome P450-dependent oxidation of MeI to formaldehyde could be detected in incubations containing hepatic or olfactory microsomes. Intact nasal turbinates were incubated with [14C]-MeI, and a dose- and time-dependent covalent binding of MeI to olfactory protein was demonstrated. The rates of protein methylation were found to be similar in OE and RE. Thus the only parameter that correlates with the site-selectivity of the observed lesion is the rate of conjugation of MeI with GSH. Whether toxicity is due to production of a reactive metabolite or GSH depletion per se, remains to be elucidated.

Animals↗
PubMed1998

The effect of a low-protein diet and dietary supplementation of threonine on tyrosine and 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione-induced corneal lesions, the extent of tyrosinemia, and the activity of enzymes involved in tyrosine catabolism in the rat.

Rats fed a low-protein diet and administered 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) orally at 30 mumol/kg/day (10 mg/kg/day) or fed a low-protein diet containing 5 ppm NTBC develop lesions to the cornea of the eye within 3-8 days of exposure with an incidence of about 80%. This treatment also produces a marked inhibition of both hepatic and renal 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, an induction of hepatic but not renal tyrosine amino transferase activity, and a marked tyrosinemia in the plasma and aqueous humor. The extent of tyrosinemia and changes in the activity of tyrosine catabolic enzymes are similar to those reported for rats fed a normal protein diet and administered NTBC orally at 30 mumol/kg/day. However, the onset of corneal lesions occurs much earlier in rats fed a low-protein diet. The adverse ocular effects of NTBC can be alleviated by supplementing the low-protein diet with 1% w/w threonine. The protection afforded by threonine inclusion in the diet was not due to any amelioration in the extent of inhibition of hepatic HPPD activity or reduction in the extent of the tyrosinemia as measured 8 days after treatment. Rats fed L-tyrosine at 5% w/w in a low-protein diet rapidly develop lesions to the cornea of the eye, which are associated with a marked tyrosinemia, increased hepatic tyrosine aminotransferase activity, and about a 50% reduction in the activity of hepatic HPPD. The onset of corneal lesions produced by feeding a high tyrosine diet could be delayed, but not prevented, by inclusion of 1% w/w threonine in the low-protein diet. The basis for the beneficial effect of dietary supplementation of threonine in alleviating the corneal lesions produced by NTBC is unclear. However, our findings do illustrate that protein deficiency limits the ability of the rat to respond to a tyrosine load produced by inhibition of HPPD.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1998

The nephrotoxicity and hepatotoxicity of 1,1,2,2-tetrafluoroethyl-L-cysteine in the rat.

Recent studies have shown that tetrafluoroethylene is a renal and hepatic carcinogen in the rat. In this study, we have examined the ability of a single i.p. dose of 1,1,2,2-tetrafluoroethyl-L-cysteine (TFEC), a major metabolite of tetrafluoroethylene, to produce hepatic and renal injury in male and female rats. We have also examined the effect of blocking the renal organic anion transport system with probenecid and of inhibiting the activity of cysteine conjugate beta-lyase with aminooxyacetic acid on the extent of renal injury produced by TFEC. Doses of > or = 12.5 mg/kg TFEC produced renal tubular necrosis to the pars recta of the proximal tubules within 24 h in both male and female rats. This was associated with an increased kidney to body weight ratio and plasma urea at doses of > or = 25 mg/kg. No consistent evidence of liver injury was seen at doses up to 50 mg/kg TFEC in rats of either sex, although occasional vacuolation of hepatocytes and a small dose-related increase in liver to body weight ratio was observed. Prior treatment of female rats with probenecid completely prevented the renal injury produced by either 25 or 50 mg/kg TFEC as judged by plasma urea and histopathology. However, prior treatment of female rats with aminooxyacetic acid afforded no protection against the nephrotoxicity produced by either TFEC or the cysteine conjugate of hexachloro-1,3-butadiene. Thus no major sex difference in nephrotoxicity in the rat was seen with TFEC, while accumulation of TFEC, or its N-acetyl derived metabolite, into renal proximal tubular cells via a probenecid sensitive transport system appears to be a key event in the mechanism of nephrotoxicity. The lack of protection observed with the cysteine conjugate beta-lyase inhibitor, aminooxyacetic acid, may reflect the inability to completely inhibit the mitochondrial form of this enzyme and thereby prevent the formation of the reactive metabolite. Our acute studies provide no insight concerning the liver carcinogenicity of tetrafluoroethylene.

Acetylcysteine↗
PubMed1998

The role of glutathione S-transferase- and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat.

The aim of this study was to investigate the role of metabolic activation in the olfactory toxicity of methyl iodide (MeI). Adult male rats were exposed via nose-only inhalation to 100 ppm MeI for 0-6 h, and non-protein sulphydryl (NP-SH) concentrations determined in selected tissues. Depletion of NP-SH occurred in all tissues, but was most marked and rapid in the respiratory epithelium of the nasal cavity and the kidney. Olfactory, lung and liver NP-SH levels were affected to a lesser extent, and those of the brain declined by only 20-30% over the whole time course. In order to modulate glutathione (GSH) status, animals were pre-treated with (1) phorone plus L-buthionine sulphoximine (BSO), which depleted NP-SH levels in all the tissues examined, or (2) the isopropyl ester of GSH (IP-GSH), which was shown to replenish NP-SH concentrations in all tissues except the liver of animals previously administered phorone. When animals were pre-treated with phorone plus BSO and then exposed to 100 ppm MeI for 2 h, there was a potentiation of the toxicity of MeI as judged by the clinical observations on the animals. In contrast, treatment with IP-GSH prior to and during exposure to MeI for 4 h afforded a marked protection to the olfactory epithelium. In order to inhibit cytochromes P450, animals were pre-treated with cobalt protoporphyrin IX. This decreased hepatic cytochrome P450 concentrations by > 90%, but when animals were then exposed to 100 ppm MeI for 4 h there was no effect on the severity of the olfactory lesion. These results indicate that conjugation of MeI with GSH is a detoxification rather than an activation pathway. Also, there is no major role for cytochrome P450-dependent oxidation in the development of the olfactory lesion.

Animals↗
PubMed1998

From toxicological problem to therapeutic use: the discovery of the mode of action of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), its toxicology and development as a drug.

NTBC is a triketone with herbicidal activity that has been shown to have a novel mode of action by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase in plants. Early studies on the toxicity of this compound found that rats treated with NTBC developed corneal lesions. Investigations aimed at understanding the mechanistic basis for the ocular toxicity discovered that the rats developed tyrosinaemia and excreted large amounts of 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate, owing to inhibition of the hepatic enzyme 4-hydroxyphenylpyruvate dioxygenase. The corneal lesions resemble those seen when rats are fed a diet supplemented with tyrosine, leading us to conclude that the ocular toxicity seen with NTBC is a consequence of a marked and sustained tyrosinaemia. Studies in collaboration with Professor Sven Lindstedt showed that NTBC was a potent inhibitor of purified human liver 4-hydroxyphenylpyruvate dioxygenase. This interaction lead to the concept of using NTBC to treat patients with tyrosinaemia type 1, to block or reduce the formation of toxic metabolites such as succinylacetoacetate in the liver. Zeneca Agrochemicals and Zeneca Pharmaceuticals made NTBC available for clinical use and, with the approval of the Swedish Medical Products Agency, a seriously ill child with an acute form of tyrosinaemia type 1 was successfully treated in February 1991. Subsequently, other children with this inborn error of metabolism in Sweden and other countries have been treated with NTBC. The drug is now available to those in need via Swedish Orphan AB.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1997

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.

Amino Acids↗
PubMed1997

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.

Administration, Oral↗
PubMed1997

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.

Animals↗
PubMed1996

Regulation of rat olfactory glutathione S-transferase expression. Investigation of sex differences, induction, and ontogenesis.

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterised with regard to sex differences, induction, and developmental regulation, and compared to those of the liver. Olfactory cytosolic GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was similar in both male and female animals, and there were no differences in subunit profile. Administration of trans-stilbene oxide and beta-naphthoflavone had no effect on olfactory GST activity with CDNB, although phenobarbitone treatment resulted in a small, but significant, increase in activity (130% compared to controls). HPLC analysis of subunit profiles indicated that all three agents induced olfactory subunit 1b and decreased subunit 6. The effect of age (3 to 84 days) on both cytosolic and microsomal CDNB activity was examined. In the liver, cytosolic activity was low at 3 days and climbed steadily to reach maximal levels around 28 days, but microsomal activity was relatively constant at all ages. Olfactory cytosolic activity was similar at all ages; microsomal activity was low until 21 days and then increased to reach a maximum at 56 days. Changes in individual cytosolic subunits were assessed by SDS-PAGE followed by immunoblotting. The significance of these results with regard to putative physiological roles for olfactory GSTs is discussed.

Aging↗
PubMed1996

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.

Amino Acids↗
PubMed1996

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.

Administration, Oral↗
PubMed1996

Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1-3-dione (NTBC): effect on enzymes involved in tyrosine catabolism and relevance to ocular toxicity in the rat.

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) to rats produced a marked tyrosinemia in the plasma and aqueous humor. The tyrosinemia was both time- and dose-dependent with the duration being more marked at the higher doses. The dose-response curve was very steep with a single dose of 1.5 micromol NTBC/kg (0.5 mg/kg) and above producing maximal concentrations of tyrosine in plasma of about 2500 nmol/ml and in aqueous humor of about 3500-4000 nmol/ml at 24 hr after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing at either 0.3 or 30 micromol/kg (0.1 or 10 mg/kg) NTBC and that the activity recovered very slowly. In response to the tyrosinemia, the activity of tyrosine aminotransferase (TAT) in the liver was induced about twofold, while the activity of homogentisic acid oxidase (HGO) was not affected. Daily oral administration of NTBC for 6 weeks induced lesions to the cornea of the eye, with a dose of 0.3 micromol/kg/day producing about a 38% incidence and a higher dose of 30 micromol/kg/day a 75% incidence. Administration of a single oral dose of [14C]NTBC at either 0.3 or 30 micromol/kg led to selective retention of radiolabel in the liver and to a lesser extent in the kidneys and the Harderian gland. Concentrations of radioactivity in the liver and kidneys remained constant over 4 days and after the lower NTBC dose were about 2 nmol/g wet wt and 0.9 nmol/g wet wt, respectively. Subcellular fractionation of the liver showed that the majority of the radiolabel, >90%, was associated reversibly with the cytosol fraction. No retention of radiolabel was detected in the cornea, the site of toxicity. Our studies indicate that NTBC binds to protein in rat liver cytosol, inhibits the hepatic cytosolic enzyme HPPD, and causes a marked and sustained tyrosinemia. We suggest that this marked and sustained ocular tyrosinemia produced by NTBC in the rat is responsible for the corneal lesions since similar corneal lesions are produced by feeding rats a high tyrosine diet.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1996

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.

Animals↗
PubMed1996

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves.

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.

Animals↗
PubMed1996

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.

Administration, Oral↗
PubMed1996

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.

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