Ibuprofen Augments Bilirubin Toxicity in Rat Cortical Neuronal Culture

Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 mu g/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 mu M unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 mu g/mL ibuprofen caused additional loss of cell viability and increased LDH release (h < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 mu M reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism. (Pediatr Res 65: 392-396, 2009)