Neurotoxic effect of L-2-chloropropionic acid on primary cultures of rat cerebellar granule cells

L-2-Chloropropionic acid (L-CPA), when administered orally to rats, produce s selective necrosis to the granule cell layer of the rat cerebellum which is delayed in onset, not appearing until 36 - 48 h after exposure. The pres ent study was conducted to characterise the toxic effect of L-CPA in primar y cell cultures of rat cerebellar granule cells in vitro. Exposure to L-CPA produced a time and concentration dependent loss in cerebellar granule cel l viability. Mean 50% effective concentration (EC50) values for L-CPA toxic ity were 18.3 +/- 0.3, 7.4 +/- 0.1, and 3.5 +/-. 0.1 mM for 24, 48 and 72 h exposure respectively. Exposure for 24 h followed by a return to L-CPA fre e medium for 24 h was more toxic than exposure for 24 h alone. Cells mainta ined in culture for a longer duration were more susceptible to L-CPA-induce d toxicity. The toxic effects of L-CPA could be partially or fully prevente d by concomitant exposure of the cells to putative neuroprotective compound s. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (3 mu M), af forded partial protection against L-CPA induced toxicity, whilst other glut amate receptor antagonists including, D(-)-2-amino-5-phosphopentanoic acid (D-APS; 300 mu M), D(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic ac id (D-CPP; 300 mu M), 5,7-dichlorokynurenic acid (10 mu M) and 6-cyano-7-ni troquinoxaline-2,3-dione (CNQX; 1 mu M) were ineffective. The antioxidant, vitamin E (10 mu M), afforded significant but incomplete protection from L- CPA toxicity. However when both MK-801 (3 mu M) and vitamin E (10 mu M) wer e present during L-CPA exposure, a greater degree of protection was observe d than with either compound alone, although the combination failed to provi de complete protection. Cyclosporin A, an inhibitor of the mitochondrial tr ansition pore, also provided partial protection. By contrast, the free radi cal trapping agent, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) prov ided concentration (1-10 mM) dependent protection against the L-CPA-induced toxicity, which was complete at 10mM. Our findings suggest that free radic al production may be involved in the mechanism of L-CPA-induced toxicity.