Bilirubin Cytotoxicity in Primary Mouse Cerebral Cortical Cell Culture

ABSTRACT

PURPOSE: We sought to quantitate the cytotoxicity of bilirubin for neuronal cells at various concentrations of bilirubin and at various [bilirubin]/[albumin] ratio. METHODS: Mouse cerebral cortical cells were obtained from 15 day-old mouse fetal cerebral cortex primary culture. Cerebral cortical cells were exposed to medium containing various concentrations of bilirubin and [bilirubin]/[albumin] ratios for 4 hours. Then, the bilirubin cytotoxicity for cerebral cortical cells was quantitated by the activity of lactate dehydrogenase (LDH) released from cerebral cortical cells into the culture media and the viability of cerebral cortical cells was quantitated by MTT (3-[4, 5 dimethylthiazol-y-yl]-2, 5-diphenyl tetrazolium bromide) activity measured 4 hours after the addition of MTT labeling reagent to the cell compartment. RESULTS: At a constant [bilirubin]/[albumin] ratio of 3: 1, the increasing concentration of bilirubin (50microM, 75microM, 100microM, 150microM) resulted in proportionally increased cytotoxicity (18+/-2%, 33+/-1%, 44+/-2%, 66+/-4%, respectively). At a constant bilirubin concentration of 86microM, the increasing [bilirubin]/[albumin] ratio (1: 3, 3: 1) also resulted in proportionally increased cytotoxicity (10+/-1%, 58+/-1%, respectively) and proportionally decreased viability (83+/-1%, 65+/-2%, respectively). CONCLUSION: Not only the concentration of bilirubin, but also the [bilirubin]/[albumin] ratio may be important for the cerebral cortial cell injury by bilirubin.