Cytotoxicity of hexachlorocyclohexane isomers and cyclodienes in primary cultures of cerebellar granule cells.

The cytotoxicity of the neurotoxic hexachlorocyclohexane (HCH) isomers alpha, beta, gamma (lindane) and delta and of the cyclodienes aldrin, endrin and alpha-endosulfan were studied in primary neuronal cultures of cerebellar granule cells. Disruption of cell membrane integrity, as indicative of cytotoxicity, was measured by propidium iodide staining. Additionally, the effects of these xenobiotic agents on three endpoints of the cellular status, concentration of intracellular-free calcium, intracellular oxygen-free radical formation and mitochondrial transmembrane potential were analyzed in parallel cultures to understand better the mechanisms of their neurocytotoxic action. To measure these parameters, the probes of the acetoxymethyl ester of fluo-3, 2',7'-dichlorofluorescin diacetate and rhodamine 123, respectively, were used. The order of cytotoxic potency of the HCH-isomers and the cyclodienes (delta-HCH > gamma-HCH > alpha-HCH approximately equal to aldrin approximately equal to alpha-endosulfan >> endrin approximately equal to beta-HCH) was very different from their in vivo LD50 order. delta-, gamma- and alpha-HCH increased the concentration of intracellular-free calcium, whereas delta- and gamma-HCH and alpha-endosulfan increased mitochondrial transmembrane potential, but none of the compounds generated oxygen-free radicals. The inhibition of delta- and gamma-HCH effects by several specific pharmacological agents suggests that delta-HCH causes its cytotoxic effects in part through intracellular Ca++ mobilization from intracellular pools sensitive to neomycin, whereas gamma-HCH acts through Ca++ influx and dantrolene-sensitive intracellular Ca++ mobilization. The use of selected endpoints of the cellular status proved to be a valuable tool to study the mechanisms of cytotoxicity.