Recovery after short-term bilirubin exposure in human NT2-N neurons

Abstract

We used human NT2-N neurons to investigate delayed effects of short-term exposure to unconjugated bilirubin (UCB). Cell viability was evaluated with MTT reduction assays and nuclear morphology. A 6-h exposure to 1, 5, or 25 μM UCB and serum deprivation (SED) significantly diminished MTT reduction. 96 h after rescue of neurons with removal of UCB and re-incubation in the original serum-containing medium, delayed effects were evident as recovery (1 μM UCB), intermediate cell death (5 μM UCB), or near complete cell death (25 μM UCB). The impact of 6 h of SED alone appeared to be modest in rescued neurons. In this model, co-treatment with the specific caspase-3 inhibitor, zDEVD.FMK (100 μM), or the pancaspase inhibitor zVAD.FMK (100 μM) did not improve viability in rescued neurons exposed to 5 μM UCB, while treatment with the NMDA receptor antagonist MK-801 (1 μM) enhanced the number of undamaged nuclei (86 ± 14% versus 50 ± 12%, P = 0.001). MK-801 had, however, no impact on MTT reduction. In a different model with a 102-h continuous exposure to UCB and SED, we found a significant additional toxic impact of serum deprivation. Separate experiments suggested that this was a result of late caspase-mediated toxicity. We conclude that UCB-mediated effects may be reversible in this model. Blockade of excitotoxic mechanisms, but not caspase activity may prevent delayed cell death.