Bilirubin‐induced cell toxicity involves PTEN activation through APE1/Ref‐1‐dependent pathway

Bilirubin‐induced neurological damage (BIND) is observed in newborns with greatly increased levels of serum unconjugated bilirubin (UCB). Early molecular events responsible for bilirubin‐induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular ROS and decreased cell survival (by the MTT test). This activated the antioxidant cell response through APE1/Ref‐1, a master redox regulator within eukaryotic cells. Activation of APE1/Ref‐1 was followed by a concomitant activation of Egr‐1 transcription factor and upregulation of PTEN tumor suppressor, an Egr‐1 target gene. Blocking ROS generation with N‐acetylcysteine pretreatment restored cell survival and limited the upregulation of PTEN in response to UCB. Cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref‐1 siRNA confirmed that UCB modulates a signaling pathway involving APE1/Ref‐1, Egr‐1 and PTEN, which may explain its toxic effects. These findings suggest new approaches for the treatment and prevention of kernicterus.