Bilirubin induces apoptosis and necrosis in human NT2-N neurons.
Studies on primary cultures of newborn rodent neurons have suggested that neuronal death induced by unconjugated bilirubin (UCB) is mainly apoptotic in nature. We exposed a human teratocarcinoma-derived cell line, NT2-N neurons, to different concentrations of UCB and albumin at a 1.5 molar ratio and used multiple, independent measures of cell damage to evaluate neuronal injury after 6, 24, and 48 h. Low doses of UCB (0.66, 2, and 5 microM) induced a moderate loss of 3-4[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) cleavage accompanied by delayed morphologic changes consistent with apoptosis (2 and 5 microM). Moderate concentrations of UCB (10 and 25 microM) resulted in early (6 h) necrosis in a subset of neurons, while remaining neurons underwent progressive impairment of MTT cleavage and increasing lactate dehydrogenase (LDH) release accompanied by predominantly delayed apoptosis. High concentrations of UCB (100 microM) induced severe impairment of MTT cleavage, extensive LDH release, and morphologic changes consistent with necrosis within 6 h. Used as a positive control for apoptosis, 2 microM STS induced progressive impairment of MTT cleavage and morphologic changes consistent with apoptosis over the entire observation period. DNA electrophoresis at 48 h was compatible with apoptosis both after treatment with STS and UCB concentrations <or=25, but not at 100 microM. Cleavage of poly (ADP-ribose) polymerase (PARP) was only seen in neurons treated with low UCB concentrations and STS. We conclude that UCB induces early necrosis at high and moderate concentrations and predominantly delayed apoptosis at low and moderate concentrations in cultured human NT2-N neurons.
Hankø
Erik
E
Department of Pediatric Research, Oslo, Norway. erikh@klinmed.uio.no
Hansen
Thor Willy Ruud
TW
Almaas
Runar
R
Lindstad
Julie
J
Rootwelt
Terje
T
D017209:Apoptosis; D001663:Bilirubin / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009336:Necrosis; D009474:Neurons / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D004396:Coloring Agents; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; D013844:Thiazoles; D004247:DNA; D007770:L-Lactate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases; C022616:thiazolyl blue; D001663:Bilirubin