Ortho-substituted but not coplanar PCBs rapidly kill cerebellar granule cells

Several PCB congeners were assessed for their cytotoxicity on cerebellar granule cells in an attempt to compare their structure-activity relationship as potential neurotoxicants and to assess the mechanisms associated with their toxicity. Flow cytometry was used to monitor the changes of a number of biochemical endpoints: membrane integrity, intracellular free calcium concentration ([Ca²⁺]_i), reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψ_m), and cell size. The non-coplanar, ortho-substituted congeners, PCB 8 (2,4′-dichlorobiphenyl), PCB 28 (2,4,4′-trichlorobiphenyl), PCB 47 (2,4,2′, 4′-tetrachlorobiphenyl), and PCB 52 (2,5,2′, 5′-tetrachlorobiphenyl) (10 μM) killed neurons to different degrees within 30 min. Loss of viability was accompanied by increased [Ca²⁺]ⁱ and decreased Δψ_m. No significant changes of ROS level were observed during exposure. The coplanar congeners, PCB 77 (3,4,3′,4′-tetrachlorobiphenyl), PCB 80 (3,5,3′,5′-tetrachlorobiphenyl), and PCB 81 (3,4,5,4′-tetrachlorobiphenyl) (10 μM), had no effects on membrane integrity, [Ca²⁺]_i or Δψ _m in this time period of exposure. In Ca²⁺-free Tyrode's medium, there was no [Ca²⁺]_i increase after exposure to the ortho-substituted congeners, but also no reduction in loss of membrane integrity, suggesting Ca²⁺ influx was not the cause of viability loss. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (1-2 μM), caused a large decrease of Δψ_m, but only a slight loss of viability, which suggested that Δψ_m is not the primary cause of PCB 52-induced cell death. These studies show that ortho-substituted PCBs are toxic to cerebellar granule cells; however, their toxic action is not secondary to elevation of intracellular calcium, a change in mitochondrial membrane potential, or free radical generation.