Výsledky projektu Studium biologického významu látek vznikajících při léčbě závažné novorozenecké žloutenky fototerapií modro-zeleným světlem

For severe unconjugated hyperbilirubinemia the gold standard treatment is
phototherapy with blue-green light, producing more polar photo-oxidation
products, believed to be non-toxic. The aim of the present study was to compare
the effects of bilirubin (BR) and lumirubin (LR), the major BR photo-oxidation product,
on metabolic and oxidative stress markers. The biological activities of these pigments
were investigated on several human and murine cell lines, with the focus on
mitochondrial respiration, substrate metabolism, reactive oxygen species
production, and the overall effects on cell viability. Compared to BR, LR was found
to be much less toxic, while still maintaining a similar antioxidant capacity in the serum
as well as suppressing activity leading to mitochondrial superoxide production.
Nevertheless, due to its lower lipophilicity, LR was less efficient in preventing
lipoperoxidation. The cytotoxicity of BR was affected by the cellular glycolytic
reserve, most compromised in human hepatoblastoma HepG2 cells. The observed
effects were correlated with changes in the production of tricarboxylic acid cycle
metabolites. Both BR and LR modulated expression of PPARα downstream effectors
involved in lipid and glucose metabolism. Proinflammatory effects of BR, evidenced by
increased expression of TNFα upon exposure to bacterial lipopolysaccharide, were
observed in murine macrophage-like RAW 264.7 cells. Collectively, these data point to
the biological effects of BR and its photo-oxidation products, which might have clinical
relevance in phototherapy-treated hyperbilirubinemic neonates and adult patients.

Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler–Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations.

Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. Methods: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. Results: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. Conclusions: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

https://doi.org/10.3390/antiox10101532