Bilirubin encephalopathy is one of the consequences of severe hyperbilirubinemia and is characterized by multifocal deposition of UCB in selected regions of the brain, resulting in temporary or permanent impairment of auditory, motor or mental functions [38
]. Cellular UCB level is closely related to the free concentration (Bf) of the pigment in plasma and to the mechanisms regulating the traffic of UCB among plasma, cerebral spinal fluid and cells. Although UCB is formed in virtually all cells and may enter by passive diffusion [39
], intracellular concentration is determined by cellular export and metabolic transformation of the pigment. Interestingly, only certain neuronal cell types present a UCB susceptibility to undergo necrosis and apoptosis [40
], whereas astrocytes are relatively spared. This has been linked to a higher content of MRP1, a transporter with high affinity to UCB [41
] able to keep the intracellular UCB concentration low. Importantly, the level of MRP1 expression in human neuroblastoma SH-SY5Y cells is inversely and linearly correlated with UCB toxic effects [16
The molecular mechanisms of intracellular UCB toxicity are still unclear [42
]. UCB has been initially proved to be a pro-apoptotic agent, suppressing cell growth by inducing DNA fragmentation, mitochondrial release of cytochrome c, activation of caspase-3 and cleavage of poly(ADP)ribose polymerase [40
]. More recently, oxidative stress has emerged as a potential crucial event, since its generation mirrors UCB-mediated apoptosis [43
]. In various cellular systems, UCB causes reactive oxygen species (ROS) production, protein oxidation, lipid peroxidation and disruption of glutathione metabolism [43
]. Furthermore, UCB-mediated oxidative stress is also in part responsible for inhibition of cell growth [48
]. Inhibition of cell proliferation has been indeed observed in primary vascular smooth muscle cells in vitro
as well as inferred by gene expression data on the repression of cell cycle-related genes in the liver of a mouse model of Crigler-Najjar type I disease [50
]. To study the effects of intracellular UCB on neurons, we took advantage of the human neuroblastoma SH-SY5Y cell line. These cells have been widely used in neurobiology as the in vitro
system of choice to dissect molecular pathways leading to neurodegeneration. Previous studies have already shown that neuroblastoma cells are a good in vitro
model to address the consequences of clinically relevant UCB concentrations [16
]. Although we are aware of the limitations in the use of a neuroblastoma cell line to recapitulate dysfunctions that occur in neonatal human brain, this experimental model allowed us to dissect in details the molecular events elicited by UCB.
Treatment with Bf 140 nM was highly toxic, triggering death in 40% of the cells within one hour. This is expected since the accepted threshold for bilirubin toxicity approximately occurs at 70 nM [5
]. 63% reduction in cell viability at 4 h after bilirubin exposure were previously demonstrated in the rat neuroblastoma N-115 cell line [7
]. Furthermore, Silva RF et al
] showed 85 nM Bf triggered deleterious effects on mitochondrial function.
A full understanding of molecular mechanisms of cell death will require additional investigation. UCB has been previously proved to induce apoptosis in neuroblastoma cells, although necrosis could not be excluded [52
Furthermore, the criteria to distinguish sensitive from insensitive neuroblastoma cells remain unclear. One may speculate that subtle heterogeneity in the level of differentiation may play a role in susceptibility to insults. Alternatively, cell death induction may depend on cell cycle position.
We then focused on the 60% of surviving cells to test the hypothesis that these neurons may express a homeostatic response.
A gene expression approach was carried out to identify gene expression patterns and signalling pathways induced upon UCB treatment. No significant changes of gene expression were observed after 1 and 4 h of UCB treatment. After 24 h, 230 genes were induced while no down-regulated genes were observed even when an FDR of 20% was applied.
The lack of an "early response" is surprising since it fails to provide a potential mechanism for cell survival within the first hour. Therefore, present data suggest that it is very unlikely that the molecular pathways induced at 24 hours play a role in the survival of cells after one hour of treatment.
GO analysis of the "late response" genes proved for the first time that UCB treatment induces an ER stress response in neuroblastoma cells in vitro
. Evidences for ER stress involvement are overwhelming. Among others, genes induced by UCB treatment include several molecular chaperones like BiP, a well-known marker of ER overload, as well as molecular components of the ERAD system, that has been recently positioned in a central stage among the molecular mechanisms of neurodegeneration [53
]. Furthermore, two well-known events in ER stress, such as XBP1
unconventional splicing and DDIT3
/CHOP nuclear relocation, were proved to be triggered by UCB treatment, therefore providing additional experimental evidences.
UCB also altered several components of the translational machinery including tRNA synthesis genes and translation initiation factors. The translation machinery is considered one of the major targets of the ER stress response to regulate protein synthesis and decrease protein overload. Since no data are available in literature about the relationship between UCB intracellular concentration and translation rate, there is a compelling need for an accurate proteomic analysis of UCB effects in SH-SY5Y neuroblastoma cells. A complementary cellular strategy in ER stress conditions includes an increase in protein degradation. In this context some UCB-induced genes suggested a role for autophagy. It is of notice that autophagy has been recently associated with neurodegenerative diseases as well as with inhibition of cell growth and entering into a quiescent state [55
]. This may correlate to previous reports showing a severe inhibition of cell proliferation mediated by UCB [49
]. Further experimental validation is needed to prove an increase of autophagy.
Finally, the induction of both subunits of the transport system X(C)(-) was also observed: SLC3A2 (4F2hc) and SLC7A11 (xCT) are able to heterodimerize to mediate cystine-glutamate exchange and regulate intracellular glutathione levels. Since the maintenance of a high glutathione concentration may be protective [57
], we speculate that induction of the X(C)(-) transport system may represent a major pro-survival response, as has been recently shown in neurons [58
]. Interestingly, we could not observe any significant differential gene expression of MRP1
at any point of the time course suggesting that this transporter plays a negligible role in UCB-induced adaptation in this in vitro