Bilirubin-Induced Oxidative Stress Leads to DNA Damage in the Cerebellum of Hyperbilirubinemic Neonatal Mice and Activates DNA Double-Strand Break Repair Pathways in Human Cells.

Vipin Rawat,Claudio Tiribelli,Andrés F Muro,Giulia Bortolussi,Silvia Gazzin

doi:10.1155/2018/1801243

Abstract

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.

Highlights

Neonatal jaundice is a frequent condition resulting mainly from the temporary delay in the activation of bilirubin conjugation in the liver [1] or from genetic mutations in the UGT1A1 gene, with the permanent loss of bilirubin conjugation activity, a condition such as the Crigler-Najjar syndromes [2]
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
In spite of the many proposed mechanisms leading to neurotoxicity during neonatal severe hyperbilirubinemia, including oxidative stress, no data are available on DNA damage in vivo and in the response mechanisms triggered by hyperbilirubinemia

Summary

Introduction

Neonatal jaundice is a frequent condition resulting mainly from the temporary delay in the activation of bilirubin conjugation in the liver [1] or from genetic mutations in the UGT1A1 gene, with the permanent loss of bilirubin conjugation activity, a condition such as the Crigler-Najjar syndromes [2]. During bilirubin-induced neurotoxicity, different molecular pathways are activated, ranging from mitochondrial damage and oxidative stress to endoplasmic reticulum (ER) stress response and inflammation [4]. Several in vitro and in vivo data demonstrated a main role of oxidative stress in cytotoxicity, when high, toxic, bilirubin concentrations are present. Incubation of synaptic vesicles, tissue culture cells, and primary cell cultures of neurons, oligodendrocytes, and astrocytes with bilirubin resulted in increases in oxidative stress and cytotoxicity [4,5,6,7,8,9,10,11,12]. In vivo studies in Gunn rats show high levels of lipid peroxidation by Oxidative Medicine and Cellular Longevity sulphadimethoxine-induced hyperbilirubinemia [13]. Studies in hyperbilirubinemic Ugt1−/− mice showed an impairment of antioxidant defenses with the activation of key oxidative stress markers [14,15,16]

Methods

Discussion

Conclusion