title: Metyrapone, an inhibitor of cytochrome oxidases, does not affect viability in a neuroblastoma cell model of bilirubin toxicity.
abstract: BACKGROUND
  Unconjugated hyperbilirubinemia may cause brain damage in infants, and globally remains a source of neonatal morbidity and mortality. A significant inter-individual variability in vulnerability to bilirubin toxicity remains largely unexplained. An enzyme located in mitochondria oxidizes bilirubin. We hypothesized that inhibiting bilirubin oxidation in human neuronal cell cultures exposed to bilirubin would increase cell death.
  
  
  METHODS
  The ability of mitochondrial membranes from CHP-212 human neuroblastoma cells to oxidize bilirubin was verified by spectrophotometry. Intact cells in culture were exposed to bilirubin (75 μM) with or without metyrapone (250 μM) for 24 h, stained with Annexin-V and Propidium iodide and analyzed for apoptosis and necrosis by flow cytometry.
  
  
  RESULTS
  Bilirubin caused a significant reduction of viability, from 84 ± 2.0% (mean ± SEM) vs 67 ± 2.7% (p < 0.05), but adding metyrapone to the bilirubin-exposed cells did not further impact cell viability. Metyrapone alone did not influence cell viability.
  
  
  CONCLUSIONS
  Herein we have shown that metyrapone does not increase cell death in neuroblastoma cells in culture exposed to bilirubin. Our results question the relationship between the oxidative mechanism evaluated by spectrophotometry and cell viability. Our findings add to the discussion on whether bilirubin oxidation represents a potentially important protective mechanism in neurons challenged by hyperbilirubinemia.
authors.affiliation: Department of Pediatric Research, Oslo University Hospital, University of Oslo, Norway.
authors.forename: Maria N
authors.initials: MN
authors.lastname: Naguib Leerberg
authors.affiliation: Department of Pediatric Research, Oslo University Hospital, University of Oslo, Norway.
authors.forename: Tomas N
authors.initials: TN
authors.lastname: Alme
authors.affiliation: Department of Neonatology, Women & Children's Division, Oslo University Hospital, Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Norway.
authors.forename: Thor W R
authors.initials: TW
authors.lastname: Hansen
keywords: Bilirubin; Cell culture; Cytochrome P450 oxidase; Kernicterus; Metyrapone; Neurotoxicity