Unconjugated
bilirubin is considered a potent
antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with
kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to
DNA, no data are available either on DNA damage in the brain triggered by
hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of
neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by
bilirubin toxicity. We studied the mechanisms associated with potential
toxic action of
bilirubin on
DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free
bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-
cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by
bilirubin, supporting the concept that DNA damage was caused by
bilirubin-induced oxidative stress.
Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair
bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and
bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with
bilirubin toxicity and may be of relevance in neonates affected with severe
hyperbilirubinemia.