Recovery after short-term bilirubin exposure in human NT2-N neurons.
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 microM 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 microM UCB), intermediate cell death (5 microM UCB), or near complete cell death (25 microM 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 microM), or the pancaspase inhibitor zVAD.FMK (100 microM) did not improve viability in rescued neurons exposed to 5 microM UCB, while treatment with the NMDA receptor antagonist MK-801 (1 microM) 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.
Hankø
Erik
E
Department of Pediatric Research, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway. erikh@ulrik.uio.no
Hansen
Thor Willy Ruud
TW
Almaas
Runar
R
Rootwelt
Terje
T
D017209:Apoptosis / Q000187:drug effects; D001663:Bilirubin / Q000633:toxicity*; D061945:Caspase Inhibitors; D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D016895:Culture Media, Serum-Free; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles
D061945:Caspase Inhibitors; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D016194:Receptors, N-Methyl-D-Aspartate; D013778:Tetrazolium Salts; D013844:Thiazoles; D016291:Dizocilpine Maleate; C022616:thiazolyl blue; D001663:Bilirubin