The Mechanism for Hexachlorocyclohexane-Induced Cytotoxicity and Changes in Intracellular Ca²⁺Homeostasis in Cultured Cerebellar Granule Neurons Is Different for the γ- and δ-Isomers

Abstract

The cytotoxic action of the δ- and γ-isomers of hexachlorocyclohexane (HCH) as well as their ability to induce changes in intracellular Ca²⁺homeostasis was studied in cultured rat cerebellar granule neurons. Changes in the free intracellular Ca²⁺concentration ([Ca²⁺]_i) related to Ca²⁺influx and release from intracellular stores were investigated 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⁺channels and release from intracellular stores, respectively. Cytotoxicity was monitored using leakage of lactate dehydrogenase (LDH) and staining 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 ester (calcein-AM). The δ-isomer of HCH was found to be more potent and active as a cytotoxic agent than the γ-isomer (lindane) and it was demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cytotoxicity induced by δ-HCH. It was noticed that pronounced (50% increase in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<25%) cells with trypan blue. Both δ- and γ-HCH induced an increase in [Ca²⁺]_iwhich was most pronounced in case of δ-HCH. Analysis of the different mechanisms governing the increase in [Ca²⁺]_iusing dantrolene, verapamil, and Ca²⁺-free medium revealed distinct differences between the two isomers with regard to the Ca²⁺pools affected. Both isomers stimulated Ca²⁺influx through voltage-gated Ca²⁺channels but only the γ-isomer affected a Ca²⁺-dependent, dantrolene-sensitive pool. On the other hand, δ-HCH affected mainly a Ca²⁺-independent dantrolene-insensitive pool. The finding that δ-HCH was more toxic than lindane may be correlated to the differences between the isomers with regard to the action on the different Ca²⁺pools. Thus, δ-HCH affected primarily the dantrolene-insensitive Ca²⁺pools which partly reflect influx. On the contrary, lindane had little effect on these Ca²⁺pools but affected primarily dantrolene-sensitive intracellular Ca²⁺stores. This may suggest that δ-HCH may exert its cytotoxic action by stimulating a large influx of Ca²⁺possibly leading to release of Ca²⁺from dantrolene-insensitive stores. In contrast, the toxic action of lindane may be primarily related to release of Ca²⁺from the dantrolene-sensitive stores.