BackgroundSevere hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo. MethodsWe used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult.ResultsWe showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKβ as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression.Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals.ConclusionsThis study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0838-1) contains supplementary material, which is available to authorized users.

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10–125 μmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.

Phototherapy was introduced in the early 1950’s, and is the primary treatment of severe neonatal jaundice or Crigler-Najjar syndrome. Nevertheless, the potential biological effects of the products generated from the photodegradation of bilirubin during phototherapy remain unknown. This is very relevant in light of recent clinical observations demonstrating that the use of aggressive phototherapy can increase morbidity or even mortality, in extremely low birthweight (ELBW) infants. The aim of our study was to investigate the effects of bilirubin, lumirubin (LR, its major photo-oxidative product), and BOX A and B (its monopyrrolic oxidative products) on the central nervous system (CNS) using in vitro and ex vivo experimental models. The effects of bilirubin photoproducts on cell viability and expression of selected genes were tested in human fibroblasts, three human CNS cell lines (neuroblastoma SH-SY5Y, microglial HMC3, and glioblastoma U-87 cell lines), and organotypic rat hippocampal slices. Neither bilirubin nor its photo-oxidative products affected cell viability in any of our models. In contrast, LR in biologically-relevant concentrations (25 μM) significantly increased gene expression of several pro-inflammatory genes as well as production of TNF-α in organotypic rat hippocampal slices. These findings might underlie the adverse outcomes observed in ELBW infants undergoing aggressive phototherapy.

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

Unconjugated bilirubin (UCB) in newborns may lead to bilirubin neurotoxicity. Few studies investigated the activation of endoplasmic reticulum stress (ER stress) by UCB. We performed an in vitro comparative study using undifferentiated SH-SY5Y, differentiated GI-ME-N neuronal cells and human U87 astrocytoma cells. ER stress and its contribution to inflammation and apoptosis induced by UCB were analyzed. Cytotoxicity, ER stress and inflammation were observed only in neuronal cells, despite intracellular UCB accumulation in all three cell types. UCB toxicity was enhanced in undifferentiated SH-SY5Y cells and correlated with a higher mRNA expression of pro-apoptotic CHOP. Mouse embryonic fibroblast knockout for CHOP and CHOP siRNA-silenced SH-SY5Y increased cells viability upon UCB exposure. In SH-SY5Y, ER stress inhibition by 4-phenylbutyric acid reduced UCB-induced apoptosis and decreased the cleaved forms of caspase-3 and PARP proteins. Reporter gene assay and PERK siRNA showed that IL-8 induction by UCB is transcriptionally regulated by NFкB and PERK signaling. These data suggest that ER stress has an important role in the UCB-induced inflammation and apoptosis, and that targeting ER stress may represent a potential therapeutic approach to decrease UCB-induced neurotoxicity.