Differential sensitivity of neural cells to bilirubin toxicity

Two established neural cell lines were used to examine the cytotoxicity of bilirubin in vitro. N2AB-1 is a subclone of Neuro 2a from the original C1300 mouse neuroblastoma and C6 glioma is a rat astrocytoma cell line. These cells were grown in monolayer cultures in medium with 10% fetal calf serum containing doses of 5 to 42 microM bilirubin. Morphologic and biochemical characteristics of cell viability were monitored for 72 h. Additional cultures of N2AB-1 were differentiated by treatment with prostaglandin E1/cyclic adenosine monophosphate before exposure to various bilirubin concentrations. The cells were examined every 24 h by phase contrast microscopy and protein synthesis was measured by incorporation of tritiated leucine for appropriate times. N2AB-1 cells were extremely sensitive to bilirubin within 24 h at doses greater than 11 microM. Cells showed swelling, vacuole formation, pigment accumulation, and lift off from the growing surface. Growth of N2AB-1 over 3 days was inhibited in a dose-dependent manner. Moreover, protein synthesis 6 h after bilirubin exposure and 24 h after bilirubin exposure was inhibited in the same dose dependency as cell growth. In contrast, N2AB-1 cells morphologically differentiated by drug treatment showed no effect of bilirubin exposure. Also, mitotically active rat glial cells were resistant to bilirubin toxicity under similar conditions. This study demonstrates that marked differences exist among neural cells in susceptibility in vitro to bilirubin toxicity and that mitotically active neuronal cells are more sensitive to bilirubin treatment than "mature" neurons.