ABSTRACT. The toxicity of bilirubin was investigated in 2 neural cell lines NBRlOA and N115 using a quantitative dye assay 3-(4,s dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [%]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and I3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 m M or higher, whereas lower bilirubin levels a t this molar ratio showed no deleterous effect. The effect of bilirubin is more pronounced a t a molar ratio of 1.5 with longer incubation periods. The M T T assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBRlOA cells, a finding which was not obtained from ['Hlthymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the M T T assay estimates the number of viable cells a t the end of the experiment by measuring mitochondria1 function whereas the [%)I]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the ['HIthymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 m M or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]-thymidine incorporation (decrease in specific activity). When the bilirubin concentration is reduced to 0.03 mM, there is little or no cell death (no change DNA) but inhibition still exists (42% decrease in specific activity).Thus, cell viability and function of these two neural lines is dependent not only on the bilirubin albumin molar ratio, but also on the absolute concentration of bilirubin and albumin as well a s the time of exposure. ciated with irreversible nuclear damage in the brain (kernicterus) (I). The toxic effect of bilirubin in tissue culture cells have been reported in a number of studies including histologic damage in dissociated rat cerebellum (2), decrease in glucose consumption in human fibroblasts (3), decrease in viability and ATP content (4), and increase in potassium leakage in modified L-929 mouse fibroblasts (5). Thaler (6) reported a decrease in DNA synthesis in hepatoma cells. Cell viability in most of these studies was assessed by the exclusion of either trypan blue or erythrocin B. The dyes will only stain those cells which are nonviable and leaky. A qualitative assessment of viable to nonviable cells is usually done by counting under the microscope.T o our knowledge, n o studies have been done on the cytotoxicity of bilirubin on neural cell lines in culture. In the present study, we evaluate the effects of bilirubin in the presence or absence of albumin on cytotoxicity in two established neural cell lines, namely N1 15, a rat neuroblastoma cell and NBRlOA a murine neuroblastoma h...