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

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.

The Comparison of Pro-Oxidant-Antioxidant Balance in Physiologic and Pathologic Jaundice

“…In the present study, pathological increase in bilirubin level, irrespective of its neurotoxic properties, activated diminishing mechanisms of oxidative stress and declined the pro-oxidantantioxidant balance of the body. This means that even a pathologic increase of bilirubin leads to an increase of antioxidants levels, which can be detected in laboratory tests; it stays in contrast to the previous studies which reported antioxidant properties only for the physiologic amounts of the bilirubin (8,24,25).…”

“…Dani et al (24) in another research stated that the antioxidant effects may be observed only in the physiologic amount of the bilirubin, and that its pathologic levels are associated with pro-oxidant effects. Similar to the mentioned articles, another investigation performed by Rawat et al (25) in neonatal mice declared that the potent antioxidant effect of the bilirubin was only observed at the moderate levels and it had pro-oxidant effects at high con-centrations. Furthermore, in contrast to our results, they attributed the neurotoxic effects of the bilirubin to oxidative stress and other mechanisms (25).…”

“…Similar to the mentioned articles, another investigation performed by Rawat et al (25) in neonatal mice declared that the potent antioxidant effect of the bilirubin was only observed at the moderate levels and it had pro-oxidant effects at high con-centrations. Furthermore, in contrast to our results, they attributed the neurotoxic effects of the bilirubin to oxidative stress and other mechanisms (25). In the present study, pathological increase in bilirubin level, irrespective of its neurotoxic properties, activated diminishing mechanisms of oxidative stress and declined the pro-oxidantantioxidant balance of the body.…”

Oxidative Stress and Inflammation in COVID-19-Associated Sepsis: The Potential Role of Anti-Oxidant Therapy in Avoiding Disease Progression

“…The symptoms mentioned above are likely brought on by low blood pressure and a hypercoagulation state by the direct and indirect action of SARS-Cov-2. Also, acidosis, high lactate, or hyperbilirubinemia have also been observed in most patients, which are all features related with oxidative stress mechanisms [ 102 , 103 ] and antioxidant responses [ 104 ]. Severe COVID-19 patients with developed typical clinical manifestations of septic shock, such as serum lactate levels >2 mmoL L −1 and persistent hypotension despite fluid resuscitation, generally require vasopressors to maintain MAP ≥ 65 mm Hg [ 105 ].…”

Microglia Susceptibility to Free Bilirubin Is Age-Dependent

“…However, we reported that concentrations of 100 and even 10 nM Bf may cause oxidative stress and injury to neocortical synaptosomes after 4 h of incubation (Brito et al, 2004). Recently, 24 h and 48 h treatment with 70 nM and 140 nM Bf compromised the cell viability of SH-SY5Y human cells and the highest concentration caused oxidative stress and DNA damage (Rawat et al, 2018). Such protective and neurotoxic distinct effects may associate to different cell types and culture models used by the researchers.…”