Flow cytometric measurements of neuronal death triggered by PCBs

We have examined the effects of several individual polychlorinated biphenyl (PCB) congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on viability and intracellular calcium concentration in acutely dissociated cerebellar granule cell neurons obtained from 7-14 day old rat pups, using a flow cytometer. Cell death was determined using the fluorescent dye, 7-aminoactinomycin-D (7-AAD), while intracellular calcium concentration was determined using Fluo-3-AM. When acutely isolated neurons were exposed to 2,4,4' trichlorobiphenyl (2,4,4', there was a dose- and time-dependent loss of viability beginning within 5 min. At a concentration of 100 microM, 80% of neurons were dead within 45 min. Loss of viability was preceded by an apparent increase in cell granularity. No significant cell death was observed upon exposure to the highly dioxin-like coplanar PCB, 3,4,5,3',4', at a concentration of 100 microM or by TCDD at a concentration of 0.1 microM. A slight loss of viability was seen with the ortho 2,6,2',6' and the coplanar 3,4,3',4', but for both it was small. All of the PCB congeners caused an early, transient increase in intracellular free calcium concentration, although TCDD did not. A small, late increase in intracellular free calcium was seen with 2,4,4', probably reflecting events leading to loss of viability. We conclude that at least one PCB congener, 2,4,4', is very neurotoxic and can cause rapid death of cerebellar granule cells.