The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the gamma- and delta-isomers.
The cytotoxic action of the delta- and gamma-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 delta-isomer of HCH was found to be more potent and active as a cytotoxic agent than the gamma-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 delta-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 delta- and gamma-HCH induced an increase in [Ca2+]i which was most pronounced in case of delta-HCH. Analysis of the different mechanisms governing the increase in [Ca2+li using 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 gamma-isomer affected a Ca2+-dependent, dantrolene-sensitive pool. On the other hand, delta-HCH affected mainly a Ca2+-independent dantrolene-insensitive pool. The finding that delta-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, delta-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 delta-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.
Rosa
R
R
Department of Pharmacology & Toxicology, Consejo Superior de Investigaciones Cientificas, Barcelona, Spain.
Sanfeliu
C
C
Suñol
C
C
Pomés
A
A
Rodríguez-Farré
E
E
Schousboe
A
A
Frandsen
A
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002492:Central Nervous System Depressants / Q000494:pharmacology / Q000633:toxicity*; D002525:Cerebellar Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D003292:Convulsants / Q000494:pharmacology / Q000633:toxicity*; D003620:Dantrolene / Q000494:pharmacology; D001556:Hexachlorocyclohexane / Q000494:pharmacology / Q000633:toxicity*; D006706:Homeostasis / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism; D007536:Isomerism; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015290:Second Messenger Systems / Q000187:drug effects*; D013329:Structure-Activity Relationship; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002492:Central Nervous System Depressants; D003292:Convulsants; C086916:delta-hexachlorocyclohexane; D001556:Hexachlorocyclohexane; D014700:Verapamil; D003620:Dantrolene; D002122:Calcium Chloride; D002118:Calcium