title: Calpain activation and not oxidative damage mediates L-2-chloropropionic acid-induced cerebellar granule cell necrosis.
abstract: 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.
authors.affiliation: Neurotoxicology Research Group, Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, United Kingdom.
authors.forename: P S
authors.initials: PS
authors.lastname: Widdowson
authors.forename: A
authors.initials: A
authors.lastname: Gyte
authors.forename: R
authors.initials: R
authors.lastname: Upton
authors.forename: J
authors.initials: J
authors.lastname: Foster
authors.forename: C T
authors.initials: CT
authors.lastname: Coutts
authors.forename: I
authors.initials: I
authors.lastname: Wyatt
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000032:analysis; D001224:Aspartic Acid / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000096:biosynthesis*; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005609:Free Radicals; D018698:Glutamic Acid / Q000187:drug effects / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D009336:Necrosis; D009422:Nervous System Diseases / Q000139:chemically induced / Q000517:prevention & control; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D011422:Propionates / Q000008:administration & dosage / Q000633:toxicity*; D051381:Rats; D013049:Spectrin / Q000032:analysis
chemical_list: D000975:Antioxidants; D005609:Free Radicals; D006843:Hydrocarbons, Chlorinated; D011422:Propionates; D013049:Spectrin; D001224:Aspartic Acid; D018698:Glutamic Acid; C021974:2-chloropropionic acid; D002154:Calpain; D001205:Ascorbic Acid