Polychlorinated biphenyls (PCB's) are a family of persistent and ubiquitous environmental contaminants which originated from heavy industrial use. There are 209 separate compounds containing variable numbers of substituted chlorine molecules. Congeners without ortho-chlorine substitutions can assume a coplanar conformation and elicit toxicity similar to dioxin. Ortho-chlorine substituted PCB's are believed to be responsible for the neurotoxicity elicited by PCB mixtures. Neurotoxic effects of PCB's include developmental delays, cognitive deficits, altered motor function, and decreased brain neuro-transmitter levels, dopamine in particular. Recently, a potential mechanism by which ortho-substituted PCB's alter dopamine content in brain and pheochromocytoma (PC12) cells has been proposed. They have been shown to elicit toxicity through calcium-selective channels, ryanodine receptors, which regulate the release of calcium from the endoplasmic reticulum (ER). Changes in intracellular calcium provide important signalling information to cells and calcium gradients across cell membranes play a key role in regulating cell-signalling in the nervous system. PCB-induced increases in calcium transport have been demonstrated to occur through direct interaction with ryanodine receptors in microsomes from mammalian brain, skeletal, and cardiac muscle, and cause increased intracellular calcium in cerebellar granule cells. The opistobranch mollusc, Aplysia, is a well-studied model of simple learning and has well-characterized neural circuits.
The aim of this study was to determine if ortho-substituted PCB's can alter intracellular calcium levels in a manner similar to that observed in mammalian cells. Aplysia bag cell neurons used were in culture for 2 days and had adhered to the surface of poly-lysine coated coverslips and formed neural processes. The cells were filled with the calcium indicator dye Fura-2/AM by bath application for 1 hour followed by three 5 minute washes with artificial sea water (ASW). Cells were imaged on the Attofluor imaging system after calibration of the system with calcium standards. 2,2',4,4'-tetrachlorobiphenyl (TCB) (100mM) was applied to the dish during imaging and images were recorded for <1 hour to observe calcium changes. Sampling rate varied from 2-60 times/minute. Isolated cells (n=3) showed increased levels of intracellular calcium after treatment with ortho-substituted PCB. The elevated calcium levels persisted and stabilized after application of TCB. One experiment was performed using five cultured cells in network. TCB (200mM) and 10 mg/ml Arocol 1254 (a PCB mixture) was applied without affecting intracellular calcium, indicating possible differences between isolated cells and cells in networks. These preliminary findings indicate that molluscs could be a good model for studying mechanisms of PCB neurotoxicity at both the cellular and behavioral levels.
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