THE MECHANISM FOR HEXACHLOROCYCLOHEXANE-INDUCED CYTOTOXICITY AND CHANGES IN INTRACELLULAR CA2-ISOMER AND DELTA-ISOMER( HOMEOSTASIS IN CULTURED CEREBELLAR GRANULE NEURONS IS DIFFERENT FOR THE GAMMA)

The cytotoxic action of the delta- and gamma-isomers of hexachlorocycl ohexane (HCH) as well as their ability to induce changes in intracellu lar Ca2+ homeostasis was studied in cultured rat cerebellar granule ne urons. Changes in the free intracellular Ca2+ concentration ([Ca2+](i) ) related to Ca2+ influx and release from intracellular stores were in vestigated using, in addition to the physiological incubation medium, media without added Ca2+ (nominally Ca2+ free) or containing verapamil or dantrolene, drugs which block influx through voltage-gated Ca2+ ch annels and release from intracellular stores, respectively. Cytotoxici ty was monitored using leakage of lactate dehydrogenase (LDH) and stai ning of damaged cells with either trypan blue or propidium iodide. In the latter case, when fluorescence microscopy was employed, undamaged cells were visualized using the vital stain calcein acetoxymethyl este r (calcein-AM). The delta-isomer of HCH was found to be more potent an d active as a cytotoxic agent than the gamma-isomer (lindane) and it w as demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cy totoxicity induced by delta-HCH. It was noticed that pronounced (50% i ncrease in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<2 5%) cells with trypan blue. Both delta- and gamma-HCH induced an incre ase in [Ca2+](i) which was most pronounced in case of delta-HCH. Analy sis of the different mechanisms governing the increase in [Ca2+](i) us ing dantrolene, verapamil, and Ca2+-free medium revealed distinct diff erences between the two isomers with regard to the Ca2+ pools affected . Both isomers stimulated Ca2+ influx through voltage-gated Ca2+ chann els but only the gamma-isomer affected a Ca2+-dependent, dantrolene-se nsitive pool, On the other hand, delta-HCH affected mainly a Ca2+-inde pendent dantrolene-insensitive pool. The finding that S-HCH was more t oxic than lindane may be correlated to the differences between the iso mers with regard to the action on the different Ca2+ pools. Thus, delt a-HCH affected primarily the dantrolene-insensitive Ca2+ pools which p artly reflect influx. On the contrary, lindane had little effect on th ese Ca2+ pools but affected primarily dantrolene-sensitive intracellul ar Ca2+ stores. This may suggest that delta-HCH may exert its cytotoxi c action by stimulating a large influx of Ca2+ possibly leading to rel ease of Ca2+ from dantrolene-insensitive stores. In contrast, the toxi c action of lindane may be primarily related to release of Ca2+ from t he dantrolene-sensitive stores. (C) 1997 Academic Press.