DNA Damage Induction by Bilirubin Induced Oxidative Stress and Activation of DNA Repair Pathways by Bilirubin

Rawat, Vipin Singh (2018). DNA Damage Induction by Bilirubin Induced Oxidative Stress and Activation of DNA Repair Pathways by Bilirubin. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000df47

Abstract

“Kernicterus” due to hyperbilirubinemia is one of main cause of irreversible brain damage in low and middle income countries. Deaths due to “kernicterus” are ranked within the top 3 causes of neonatal death in African countries. Neonates experiencing permanent or temporary absence or reduced activity of Uridine diphosphate-glucuronosyl-transferase A1 (UGT1A1) enzyme present increased levels of total unconjugated bilirubin (UCB) in the blood. When the bilirubin binding capacity of serum albumin is saturated, this results in the increase in the free fraction of bilirubin (Bf). The excess of Bf is accumulated in lipid-rich tissues such as the brain, where it may reach toxic levels causing bilirubin-induced encephalopathy in jaundiced newborns and patients with Crigler-Najjar syndrome Type I, leading to kernicterus if not promptly treated. Bilirubin causes severe neurological dysfunction by affecting several different cellular pathways such as, induction of oxidative stress, endoplasmic reticulum (ER) stress, autophagy, neuroinflammation and DNA damage.

The work done in this thesis focus on the DNA damaging effect of bilirubin on neuronal (SH SY 5Y) and non-neuronal (HeLa) cells and the effect of bilirubin on the different double stranded break (DSB) repair pathways was investigated using HeLa DR GFP and HeLa cell lines.

Bilirubin exposure led to time-dependent increase in DNA damage in neuronal cells. Treatment with the anti-oxidant N-acetylcysteine (NAC) caused a dose-dependent decrease in DNA damage, suggesting a key role of oxidative stress in bilirubin-induced DNA damage.

In the second part of this work, I studied the effect of bilirubin on different DSB repair pathways using HeLa cell lines. Treatment with bilirubin modulated Homologous Recombination (HR) in a time-dependent manner. In fact, treatment with different doses of bilirubin led to a dose-dependent increase in HR. Bilirubin-induced increase in HR was reversed by addition of NAC, suggesting the important role of oxidative stress in bilirubin-induced modulation of HR. DNA damage analysis after bilirubin treatment showed an increase in DNA damage in HeLa cells. Unexpectedly, bilirubin-induced DNA damage had no effect on the cell cycle profile. Similarly, exposure to bilirubin concentrations similar to those found in patients led to an increase in the Non-Homologous End Joining (NHEJ) pathway. These results suggest the general increase in DSB repair pathways by bilirubin.

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