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Bilirubin-induced neurologic dysfunction (BIND) and classical kernicterus are clinical manifestations of moderate to severe hyperbilirubinemia whenever bilirubin levels exceed the capacity of the brain defensive mechanisms in preventing its entrance and cytotoxicity. In such circumstances and depending on the associated co-morbidities, bilirubin accumulation may lead to short- or long-term neurodevelopmental disabilities, which may include deficits in auditory, cognitive, and motor processing. Neuronal cell death, astrocytic reactivity, and microglia activation are part of the bilirubin-induced pathogenesis. Less understood is how abnormal growth and maturation of oligodendrocytes may impact on brain development, affecting the formation of myelin tracts. Based on in-vitro and in-vivo models, as well as in clinical cases presented here, we propose the existence of impaired myelination by bilirubin with long-term sequelae, mainly in pre-term infants. Sensitive time-windows are highlighted and centered on the different developmental-dependent impairments determined by bilirubin, and the influence of sepsis and hypoxia is reviewed.

The blood–brain barrier (BBB) is a complex and dynamic structure that plays a key role in central nervous system (CNS) homeostasis. It strictly regulates the entrance of molecules into the brain parenchyma and prevents the access of neurotoxins and pathogens while promoting the efflux of several molecules. The brain microvascular endothelial cells are the anatomical basis of the BBB, which has unique characteristics such as the elaborate junctional complexes that nearly obliterate the intercellular space as well as the presence of influx and efflux transporters. Endothelial cells establish important interactions with glial cells, neurons, and perivascular pericytes as well as with the acellular components of the basement membrane, which together constitute the neurovascular unit. BBB disruption has been reported in a wide range of CNS pathologies, with an emerging role in the onset and disease progression. Accordingly, recent studies revealed vascular dysfunction in neonatal jaundice, a common pathology in the early neonatal period affecting 1/10 children presenting values of total bilirubin >17 mg/dL (291 μM). Here we summarize the clinical aspects of moderate to severe neonatal jaundice and provide a comprehensive review of the literature regarding bilirubin-induced neurotoxicity from a vascular-centered approach. The collected evidence place endothelial dysfunction and pericyte demise as key players in the disruption of CNS homeostasis, mainly in cases of lasting hyperbilirubinemia, thus pointing to novel targets to prevent neurological dysfunction due to severe neonatal jaundice.

Research on the mechanisms of bilirubin-induced neurological dysfunction focuses mainly on neuronal death, astrocyte-mediated events and microglia activation. Although myelin damage by unconjugated bilirubin (UCB) has been documented in neonatal kernicterus cases, the events leading to myelination impairment were never explored. This condition may occur by reduced oligodendrocyte precursor cells (OPC) number, or failure of OPC to differentiate in myelinating oligodendrocytes. We have shown that UCB elicits an inflammatory response, glutamate release and reactive oxygen species (ROS) generation in neurons and glial cells, biomolecules with toxic properties on OPC. Hence, we propose to examine whether UCB determines OPC demise and, if so, which signaling pathways are involved. Our results show that OPC display increased apoptosis and necrosis-like cell death upon UCB exposure, mediated by early signals of endoplasmic reticulum (ER) stress [e.g. upregulation of glucose-regulated protein (GRP)78, inositol-requiring enzyme (IRE)-1α and activation transcription factor (ATF)-6, as well as activation of caspase-2 and c-Jun N-terminal kinase (JNK)], followed by mitochondrial dysfunction (e.g. loss of mitochondria membrane potential and caspase-9 activation). The later calpain activation points to intracellular Ca(2+) overload and intervention of both ER and mitochondria. Downstream production of ROS may derive from mitochondria damage and secondary injuries, possibly determining the second cycle of GRP78, IRE-1α, caspase-2 and JNK activation. Moreover, inhibition of caspases, calpains and oxidative stress, by using specific inhibitors, prevented UCB-induced OPC death. UCB did not induce the release of cytokines or glutamate by OPC. These results indicate that UCB by reducing OPC survival, through a cascade of programmed intracellular events triggered by ER stress and mitochondria dysfunction, can compromise myelinogenesis.

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.

Encephalopathy by hyperbilirubinemia in infants has been described for decades, but neither the underlying cellular and molecular mechanisms nor the selective pattern of bilirubin deposition in the brain is well understood. The brain is composed of highly specialized and diverse populations of cells, represented by neurons and glia that comprise astrocytes, oligodendrocytes, and microglia. Although microscopic evaluation of icteric brain sections revealed bilirubin within neurons, neuronal processes, and microglia, cell dependent-sensitivity to bilirubin toxicity and the role of each nerve cell type are poorly understood. Even less considered are glial and neuronal pathologic alterations as integrated phenomena. The available knowledge on reactivity of glial cells to bilirubin and on the impairment to neuronal network dynamics that it causes, here summarized, suggests that a better comprehension of the interplay between neurons and glia is essential to understand bilirubin neurotoxicity and highlight potential molecular targets that may lead to disease-modifying therapeutic approaches.

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 µM UCB, in the presence of 100 µM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 µM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of β-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and β-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.

Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1β signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNF-α receptor (TNFR)1 and IL-1β receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1β cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets.

High levels of unconjugated bilirubin (UCB) may initiate encephalopathy in neonatal life, mainly in pre-mature infants. The molecular mechanisms of this bilirubin-induced neurologic dysfunction (BIND) are not yet clarified and no neuroprotective strategy is currently worldwide accepted. Here, we show that UCB, at conditions mimicking those of hyperbilirubinemic newborns (50 microM UCB in the presence of 100 muM human serum albumin), rapidly (within 1 h) inhibited cytochrome c oxidase activity and ascorbate-driven oxygen consumption in 3 days in vitro rat cortical neurons. This was accompanied by a bioenergetic and oxidative crisis, and apoptotic cell death, as judged by the collapse of the inner-mitochondrial membrane potential, increased glycolytic activity, superoxide anion radical production, and ATP release, as well as disruption of glutathione redox status. Furthermore, the antioxidant compound glycoursodeoxycholic acid (GUDCA) fully abrogated UCB-induced cytochrome c oxidase inhibition and significantly prevented oxidative stress, metabolic alterations, and cell demise. These results suggest that the neurotoxicity associated with neonatal bilirubin-induced encephalopathy occur through a dysregulation of energy metabolism, and supports the notion that GUDCA may be useful in the treatment of BIND.

It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.

We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01-1.0 microM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with N-acetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage.

Microglia are the resident immune cells of the brain and are the principal source of cytokines produced during central nervous system inflammation. We have previously shown that increased levels of unconjugated bilirubin (UCB), which can be detrimental to the central nervous system during neonatal life, induce the secretion of inflammatory cytokines and glutamate by astrocytes. Nevertheless, the effect of UCB on microglia has never been investigated. Hence, the main goal of the present study was to evaluate whether UCB leads to microglial activation and to the release of the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. Additionally, we investigated the effects of UCB on glutamate efflux and cell death. The results showed that UCB induces morphological changes characteristic of activated microglia and the release of high levels of TNF-alpha, IL-1beta, and IL-6 in a concentration-dependent manner. In addition, UCB triggered extracellular accumulation of glutamate and an increased cell death by apoptosis and necrosis. These results demonstrate, for the first time, that UCB is toxic to microglial cells and point to microglia as an important target of UCB in the central nervous system. Moreover, they suggest that UCB-induced cytokine production, by mediating cell injury, can further contribute to exacerbate neurototoxicity. Interestingly, microglia cells are much more responsive to UCB than astrocytes. Collectively, these data indicate that microglia may play an important role in the pathogenesis of encephalopathy during severe hyperbilirubinemia.

Unconjugated bilirubin (UCB) encephalopathy is a predominantly early life condition resulting from the impairment of several cellular functions in the brain of severely jaundiced infants. However, only few data exist on the age-dependent effects of UCB and their association with increased vulnerability of premature newborns, particularly in a sepsis condition. We investigated cell death, glutamate efflux, and inflammatory cytokine dynamics after exposure of astrocytes at different stages of differentiation to clinically relevant concentrations of UCB and/or lipopolysaccharide (LPS). Younger astrocytes were more prone to UCB-induced cell death, glutamate efflux, and inflammatory response than older ones. Furthermore, in immature cells, LPS exacerbated UCB effects, such as cell death by necrosis. These findings provide a basis for the increased susceptibility of premature newborns to UCB deleterious effects, namely when associated with sepsis, and underline how crucial the course of cell maturation can be to UCB encephalopathy during moderate to severe neonatal jaundice.

Unconjugated bilirubin (UCB), at slightly elevated unbound concentrations, is toxic to astrocytes and neurons, damaging mitochondria (causing impaired energy metabolism and apoptosis) and plasma membranes (causing oxidative damage and disrupting transport of neurotransmitters). Accumulation of UCB in the CSF and CNS is limited by its active export, probably mediated by MRP1/Mrp1 present in choroid plexus epithelia, capillary endothelia, astrocytes and neurons. Upregulation of MRP1/Mrp1 protein levels by UCB might represent an important adaptive mechanism that protects the CNS from UCB toxicity. These concepts could explain the varied susceptibility of newborns to bilirubin neurotoxicity and the occurrence of neurological damage at plasma UCB concentrations well below therapeutic guidelines, and are relevant to the increasing prevalence of bilirubin encephalopathy in newborns.

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0–8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0–7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.

Increased levels of unconjugated bilirubin, the end-product of heme catabolism, are detrimental to the central nervous system. To examine the role of apoptosis in bilirubin-induced toxicity and to characterize the biochemical pathway of cell death, we exposed developing rat brain neurons to purified unconjugated bilirubin at concentrations below and above saturation of human serum albumin. Isolated neurons treated with bilirubin showed increased levels of apoptosis. Mitochondrial cytochrome c was extensively released and accumulated in cytosol. Consistent with this observation, caspase-3 was activated and the full-length substrate poly(ADP)ribose polymerase (PARP) degraded, even in the presence of very modestly elevated concentrations of bilirubin. In parallel, all events were prevented in cells preincubated with ursodeoxycholate. Further experiments showed that bilirubin diminished mitochondrial transmembrane potential (DeltaPsi(m)) and increased mitochondrial-associated Bax protein levels, while directly disrupting membrane lipid and protein structure. In conclusion, bilirubin induces mitochondrial depolarization and Bax translocation via physical interaction with membranes, mediating the mitochondrial pathway of apoptosis in neurons exposed to bilirubin. These results provide a novel insight into the mechanism of bilirubin-induced toxicity.

High levels of unconjugated bilirubin (UCB) can be neurotoxic. Nevertheless, the mechanism of UCB interaction with neural cells is still unknown. This study investigates whether cultured rat neurons and astrocytes respond differently to UCB exposure. UCB toxicity was evaluated by lactate dehydrogenase release, induction of apoptosis, cytoskeleton degeneration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and glutamate uptake. Primary cultures of rat brain astrocytes and neurons were incubated at 37 degrees C with 85.5 microM UCB plus 28.5 microM albumin for 4 h. In assays of glutamate uptake, cells were exposed to 80-120 microM UCB plus 100 microM albumin for 15 min. The results showed that after incubation with 85.5 microM UCB, lactate dehydrogenase release was greater in neurons than in astrocytes (38% versus 14%, p < 0.05). Also, levels of apoptosis were markedly enhanced in neurons (29% versus 19%, p < 0.01). In accordance, neuronal cytoskeleton disassembly was evident during incubation with 85.5 microM UCB, whereas equivalent effects on astrocytes required as much as 171 microM. Conversely, inhibition of MTT metabolism and glutamate uptake by UCB was more pronounced in astrocytes than in neurons (74% versus 60%, p < 0.05 and 41% to 56% versus 25% to 33%, p < 0.05, respectively). In conclusion, the study demonstrates that astrocytes are more susceptible to inhibition of glutamate uptake and MTT reduction by UCB, whereas neurons are more sensitive to cell death by necrosis or apoptosis. These results suggest that UCB is toxic to both astrocytes and neurons, although through distinct pathways.

The pathogenesis of bilirubin encephalopathy appears to result from accumulation of unconjugated bilirubin (UCB), which, in turn, may cause mitochondrial perturbation, release of intermembrane proteins, and, ultimately, cell death. Aging imparts to cells a different susceptibility to this toxic stimulus, as neonates are particularly vulnerable to the accumulation of UCB in the CNS. In this paper, we further characterize UCB-induced toxicity in isolated neuronal and glial cells according to age in culture. In addition, we investigate sensitivity of mitochondria derived from young and old rats to UCB-induced membrane permeabilization and, finally, evaluate whether age-dependent changes in UCB toxicity are accompanied by alterations in the mitochondrial content of cytochrome c. The results showed that UCB is more toxic to immature neural cells after 4-5 d in culture (p < 0.001), whereas neurons were more sensitive than astrocytes (p < 0.05). In fact, approximately 40% of cells were apoptotic in immature cultures compared with 20% in mature cultures. Unexpectedly, mitochondrial swelling and subsequent efflux of cytochrome c induced by UCB were 2-fold greater in organelles derived from older rats (p < 0.01). In conclusion, UCB toxicity of isolated rat neuronal and glial cells is modulated by age in culture in that immature cells are more susceptible. Mitochondria derived from younger rats are nevertheless more resistant to membrane permeabilization and cytochrome c release induced by UCB. The data indicate that the cells of young animals are relatively resistant to UCB toxicity, through a protective mechanism at the mitochondrial level; however, this is not sufficient to prevent apoptosis of cells in the young animal. Thus, although playing a role, direct mitochondrial injury may not be the sole mechanism of UCB cytotoxicity.

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 micromol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37 degrees C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p0.05), and in elution of membrane cholesterol (73%, p0.01) and phospholipids (123%, p0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (-36%, p0.01), as well as that of phospholipids by TFP (-28%, p0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.

Background Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. Materials and methods Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 8 Co r 378C, after 4 h incubation at pH 7·4, with increasing molar ratios of bilirubin to albumin (0·5‐5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. Results Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 8 Cv s. 48C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P< 0·01). The loss of membrane lipids was remarkable at molar ratios $ 0·5, both at 4 8C and 37 8 C( P< 0·01), although correlation with bilirubin concentration was only significant at 37 8 C( ra 0·971; P< 0·01). Conclusions These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocytebound bilirubin during severe hyperbilirubinaemia.

The cerebellum is one of the most affected brain regions in the course of bilirubin-induced neurological dysfunction. We recently demonstrated that unconjugated bilirubin (UCB) reduces oligodendrocyte progenitor cell (OPC) survival and impairs oligodendrocyte (OL) differentiation and myelination in co-cultures of dorsal root ganglia neurons and OL. Here, we used organotypic cerebellar slice cultures, which replicate many aspects of the in vivo system, to dissect myelination defects by UCB in the presence of neuroimmune-related glial cells. Our results demonstrate that treatment of cerebellar slices with UCB reduces the number of myelinated fibres and myelin basic protein mRNA expression. Interestingly, UCB addition to slices increased the percentage of OPC and decreased mature OL content, whereas it decreased Olig1 and increased Olig2 mRNA expression. These UCB effects were associated with enhanced gliosis, revealed by an increased burden of both microglia and astrocytes. Additionally, UCB treatment led to a marked increase of tumor necrosis factor (TNF)-α and glutamate release, in parallel with a decrease of interleukin (IL)-6. No changes were observed relatively to IL-1β and S100B secretion. Curiously, both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and TNF-α antibody partially prevented the myelination defects that followed UCB exposure. These data point to a detrimental role of UCB in OL maturation and myelination together with astrocytosis, microgliosis, and both inflammatory and excitotoxic responses, which collectively may account for myelin deficits following moderate to severe neonatal jaundice.

Morphology and membrane lipid composition of erythrocytes from neonates (jaundiced and healthy) and adults (before and after incubation with bilirubin) were studied. The morphological index, expressing the relative proportions of the different stages of cell distortion, and the membrane cholesterol, phospholipids and cholesterol/phospholipids molar ratio, were determined. In jaundiced neonates a significant increase in the morphological index (P < 0.01) was found. After incubation with bilirubin, adult erythrocytes also showed an increase in the morphological index (P < 0.01). Hemolysis occurred under these conditions, and the red cell ghosts obtained (vesicles) showed a rounded morphology. Higher cholesterol/phospholipid ratio and lower phospholipid content were found in jaundiced neonates compared with healthy babies (P < 0.05) and adults (P < 0.01), as well as in the cells (P < 0.05) and vesicles (P < 0.01) obtained after bilirubin incubation. Bilirubin cytotoxicity may occur in a stepwise manne: deposition of bilirubin in membrane produces echinocytosis, which is followed by disintegration of the lipid bilayer with loss of phospholipids from the membrane.

Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation, and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis, cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-α secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. This condition may lead to the deposition of unconjugated bilirubin (UCB) in the central nervous system, causing nerve cell damage by molecular and cellular mechanisms that are still being clarified. To date, all the studies regarding bilirubin-induced neurological dysfunction were performed in monotypic nerve cell cultures. The use of co-cultures, where astrocyte-containing culture inserts are placed on the top of neuron cultures, provides the means to directly evaluate the cross-talk between these two different cell types. Therefore, this study was designed to evaluate whether protective or detrimental effects are produced by astrocytes over UCB-induced neurodegeneration. Our experimental model used an indirect co-culture system where neuron-to-astrocyte signaling was established concomitantly with the 24 h exposure to UCB. In this model astrocytes abrogated the well-known UCB-induced neurotoxic effects by preventing the loss of cell viability, dysfunction and death by apoptosis, as well as the impairment of neuritic outgrowth. To this protection it may have accounted the induced expression of the multidrug resistance-associated protein 1 and the 3.5-fold increase in the values of S100B, when communication between both cells was established independently of UCB presence. In addition, the presence of astrocytes in the neuronal environment preserved the UCB-induced increase in glutamate levels, but raised the basal concentrations of nitric oxide and TNF-α although no UCB effects were noticed. Our data suggest that bidirectional signalling during astrocyte-neuron recognition exerts pro-survival effects, stimulates neuritogenesis and sustains neuronal homeostasis, thus protecting cells from the immediate UCB injury. These findings may help explain why irreversible brain damage usually develops only after the first day of post-natal life.

BackgroundSepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC).Methodology/principal findingsMonolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB.ConclusionsLPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

Neuronal oxidative damage and cell death by unconjugated bilirubin (UCB) showed to be mediated by overstimulation of glutamate receptors and nitric oxide (NO) production, which was abrogated by the bile acid glycoursodeoxycholic acid (GUDCA). Microglia, a crucial mediator of CNS inflammation, evidenced to react to UCB by releasing glutamate and NO before becoming senescent. Our studies demonstrated that neurite outgrowth deficits are produced in neurons exposed to UCB and that conditioned media from these UCB-treated neurons further stimulate NO production by microglia. Nevertheless, microglia protective and/or harmful effects in neonatal jaundice are poorly understood, or unrecognized. Here, we investigated the role of microglia, glutamate and NO in the impairment of neurite sprouting by UCB. Therapeutic potential of the anti-inflammatory cytokine interleukin (IL)-10 and GUDCA was also evaluated. By using MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific NO synthase inhibitor) we found that glutamate and NO are determinants in the early and enduring deficits in neurite extension and ramification induced by UCB. Both GUDCA and IL-10 prevented these effects and decreased the production of glutamate and NO. Only GUDCA was able to counteract neuronal death and synaptic changes. Data from organotypic-cultured hippocampal slices, depleted or non-depleted in microglia, supported that microglia participate in glutamate homeostasis and contribute to NO production and cell demise, which were again abrogated by GUDCA. Collectively our data suggest that microglia is a key player in UCB-induced neurotoxicity and that GUDCA might be a valuable preventive therapy in neonates at risk of UCB encephalopathy.

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1β promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.

Hyperbilirubinemia is a common condition in neonatal life, where elevated levels of unconjugated bilirubin (UCB) may lead to adverse neurologic outcomes, namely in the presence of inflammatory features. In this review, we summarize recent concepts on UCB damage to brain cells and associated neuroinflammation research. Exposure of astrocytes and microglia to UCB initiates an inflammatory response with the release of proinflammatory cytokines, such as TNF-alpha, IL-1beta and IL-6, accumulation of extracellular glutamate and a time-dependent cell death. Moreover, undifferentiated cells revealed to be particularly susceptible to UCB-induced immunostimulation pointing to a mechanism that may preside to the vulnerability evidenced by premature newborns. Evaluation of intracellular mechanisms of astrocyte and microglia to UCB revealed that TNF-alpha and IL-1beta pathways as well as MAPK and NF-kappaB signaling cascades are key mediators of both cytokine production and cell toxicity observed upon UCB challenge. Understanding these mechanisms is essential for the development of new strategies targeting UCB-induced neurotoxicity. Thus, a therapeutic approach for the prevention or amelioration of neurological deficits resulting from moderate to severe hyperbilirubinemia, may consist on the use of immunomodulators, such as IL-10 that showed ability to suppress the release of cytokines from astrocytes exposed to UCB, glycoursodeoxycholic acid (GUDCA) that abrogated both UCB-stimulated cytokine secretion and UCB-induced loss of cell survival, and minocycline that evidenced a unique role in preventing neurodegeneration in in vitro and in vivo models. Novel pharmacological strategies may reduce the incidence of UCB encephalopathy and prevent minor cerebral lesions that may result in mental illness.

Unconjugated bilirubin (UCB) injury to glial cells leads to the secretion of glutamate and elicits a typical inflammatory response. Release of pro-inflammatory cytokines may influence gliogenesis and neurogenesis, and lead to deficits in learning and memory. Glutamate metabolism dysregulation and overexpression of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta are consistent with schizophrenia neuropathology. Recently, an increased prevalence of schizophrenia was reported in individuals with Gilbert's syndrome and among those who have had elevated levels of UCB in the neonatal life. In this review, we explore the reactivity of astrocytes, neurons and microglia to UCB, the cascade of events implicated in the immunostimulant effects of UCB, as well as the role of each nerve cell type and maturation state in the neuropathology of UCB. Identification of the signaling events promoted by UCB will be relevant for developing novel therapies that might reduce the risk of brain injury and disabilities.

Elevated levels of serum unconjugated bilirubin (UCB) in the first weeks of life may lead to long-term neurologic impairment. We previously reported that an early exposure of developing neurons to UCB, in conditions mimicking moderate to severe neonatal jaundice, leads to neuritic atrophy and cell death. Here, we have further analyzed the effect of UCB on nerve cell differentiation and neuronal development, addressing how UCB may affect the viability of undifferentiated neural precursor cells and their fate decisions, as well as the development of hippocampal neurons in terms of dendritic and axonal elongation and branching, the axonal growth cone morphology, and the establishment of dendritic spines and synapses. Our results indicate that UCB reduces the viability of proliferating neural precursors, decreases neurogenesis without affecting astrogliogenesis, and increases cellular dysfunction in differentiating cells. In addition, an early exposure of neurons to UCB decreases the number of dendritic and axonal branches at 3 and 9 days in vitro (DIV), and a higher number of neurons showed a smaller growth cone area. UCB-treated neurons also reveal a decreased density of dendritic spines and synapses at 21 DIV. Such deleterious role of UCB in neuronal differentiation, development, and plasticity may compromise the performance of the brain in later life.

Nerve cell injury induced by unconjugated bilirubin (UCB) has been implicated in brain damage during severe neonatal hyperbilirubinemia, although the molecular mechanisms underlying UCB neurotoxicity are still not clarified. It has been suggested recently that there is an association between hyperbilirubinemia and long-term neurologic dysfunctions. We incubated immature neurons with UCB to evaluate the short- and long-term effects of UCB on apoptotic death and on neuritic outgrowth and ramification. We also evaluated whether mature neurons, exposed previously to UCB in an early stage of differentiation, are more sensitive to apoptosis or to neuritic breakdown when treated with inflammatory agents, such as lipopolysaccharide and tumor necrosis factor-alpha. Results show that exposure of immature neurons to UCB increased apoptosis and provoked a reduction of both neurite extension and number of nodes. These injurious effects observed in immature cells treated with UCB were increasingly perpetuated along cell differentiation, as compared to neurons incubated in the absence of UCB. In addition, neurons that were exposed to UCB when immature showed an increased susceptibility to death by apoptosis, as well as an additional decrease in neurite outgrowth when incubated with an inflammatory agent afterward. This work shows, for the first time, that UCB induces neurite changes consistent with neurodevelopment abnormalities. Furthermore, pre-exposure to UCB followed by an inflammatory stimulus leads to an enhanced susceptibility to long-term apoptosis, as well as a greater neuritic breakdown. These data support the association between neonatal hyperbilirubinemia and the later development of mental illness, such as schizophrenia.

When activated by unconjugated bilirubin (UCB), astrocytes are important sources of inflammatory mediators such as TNF-alpha, IL-1beta and IL-6, which may contribute for the neurotoxicity observed during severe neonatal hyperbilirubinemia. In the present study, we have addressed the role of the mitogen-activated protein kinases (MAPKs) p38, Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)1/2 pathways and their relation with the nuclear factor kappaB (NF-kappaB) cascade in the signalling events involved in cytokine release and cell death caused by UCB in primary cultures of rat astrocytes. Stimulation of astrocytes with UCB in the presence of all the MAPK inhibitors prevented UCB-induced release of TNF-alpha and IL-6, while IL-1beta secretion was only reduced by JNK1/2 and ERK1/2 inhibitors. In addition, activation of the NF-kappaB transcription factor, needed for cytokine release by UCB-stimulated astrocytes, was shown to be dependent on JNK1/2 and ERK1/2 phosphorylation. Moreover, all MAPK inhibitors prevented astroglial apoptosis triggered by UCB. Interestingly, UCB-induced lactate dehydrogenase release was prevented by blockade of JNK1/2, ERK1/2 and NF-kappaB cascades but enhanced by p38 inhibition. Taken together, our data demonstrate for the first time that MAPK transduction pathways are key players in the UCB-induced inflammatory response and cell death in astrocytes, probably also involving NF-kappaB modulation. These findings contribute to unraveling the complex mechanisms of astrocyte reactivity to UCB and may ultimately prove useful in the development of new therapeutic strategies to prevent nerve cell damage during acute bilirubin encephalopathy.

The pathogenesis of bilirubin encephalopathy seems to result from accumulation of unconjugated bilirubin (UCB) within the brain. We have recently demonstrated that UCB causes astroglial release of proinflammatory cytokines and glutamate, as well as cell death. The bile acid glycoursodeoxycholic acid (GUDCA) and the anti-inflammatory cytokine interleukin (IL)-10 have been reported to modulate inflammation and cell survival. In this study we investigated the effect of these therapeutic agents on the astroglial response to UCB. Only GUDCA prevented UCB-induced astroglial death. The secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta elicited by UCB in astrocytes was reduced in the presence of GUDCA and IL-10, whereas the suppression of IL-6 was only counteracted by GUDCA. Neither GUDCA nor IL-10 modulated the accumulation of extracellular glutamate. Additionally, IL-10 markedly inhibited UCB-induced nuclear factor-kappaB nuclear translocation and cytokine mRNA expression, whereas GUDCA only prevented TNF-alpha mRNA expression. Moreover, GUDCA inhibited TNF-alpha- and IL-1beta-converting enzymes, preventing the maturation of these cytokines and their consequent release. Collectively, this study shows that IL-10 action is restricted to UCB-induced release of TNF-alpha and IL-1beta from the astrocytes, whereas GUDCA presents a more ubiquitous action on the astroglial reactivity to UCB. Hence, GUDCA may have potential benefits over an IL-10 therapeutic approach in reducing UCB-induced astrocyte immunostimulation and death.

Hypoxia–ischemia in the perinatal period is a common cause of neurologic disability in children and is often associated with neonatal morbidity and mortality. Another frequent condition of the newborn is hyperbilirubinemia and it is well known that deposition of unconjugated bilirubin (UCB) in the central nervous system can damage nerve cells and cause encephalopathy. Interestingly, some studies report the onset of cerebral hypoxia–ischemia as a risk factor for UCB encephalopathy, since that condition often precedes neonatal hyperbilirubinemia. However, the cellular mechanisms triggered by hypoxia–ischemia that may enforce UCB deleterious effects are not well elucidated. Therefore, we designed this study to investigate whether hypoxia (HP) or combined oxygen–glucose deprivation (OGD) followed by reoxygenation, modifies glial cell susceptibility to UCB injury. Thus, cultured astrocytes were exposed to HP or OGD for 4 h and returned to normoxic conditions for another 12 h prior to incubation with UCB for 4 h. HP and OGD effects in UCB toxicity were compared to normoxic conditions. Our results demonstrate that HP and OGD preconditioning increase the vulnerability of glial cells to UCB damage by enhancing some of the deleterious effects of UCB, namely cell death by both apoptosis and necrosis. This preconditioning also augments the UCB-induced stimulation of an inflammatory response by an effect that involves the activation of the nuclear factor κB activation. These findings provide a novel basis for the increased risk of brain damage in jaundiced newborns that were previously exposed to hypoxia or ischemia during the perinatal period, namely during delivery.

Nerve cell injury by unconjugated bilirubin (UCB) has been implicated in brain damage during neonatal hyper- bilirubinemia, particularly in the preterm newborn. Recently, it was shown that UCB is a substrate for the multidrug resis- tance-associated protein 1 (Mrp1), an ATP-dependent efflux pump, which may decrease UCB intracellular levels. To ob- tain a further insight into the role of Mrp1 in the increased vulnerability of immature cells to UCB, we evaluated the mRNA and the protein levels of Mrp1 throughout differentia- tion in primary cultures of rat neurons and astrocytes. Fur- thermore, in order to provide supportive evidence for the role of Mrp1 in the protection of nerve cells from UCB-induced effects, we evaluated cell susceptibility to UCB when Mrp1 was inhibited with MK571 ((E)-3-(((3-(2-(7-chloro-2-quinolinyl) ethenyl)phenyl)-((3-dimethylamino)-3-oxopropyl)thio)methyl)- thio)-propanoic acid). The results are the first to demonstrate that Mrp1 is expressed in neurons and that both mRNA and protein levels of Mrp1 increase with cell differentiation. Addi- tionally, inhibition of Mrp1 was associated with an increase in UCB toxic effects, namely cell death, cell dysfunction, and se- cretion of interleukin (IL)-1, tumor necrosis factor (TNF)- ,a s well as of glutamate. These results point to a novel role of Mrp1 in the susceptibility of premature babies to UCB encephalopa- thy, and provide a startup point for the development of a new therapeutic strategy. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

Neonatal jaundice, a physiologic condition reflecting the interplay between developmentally modulated changes in bilirubin production and metabolism, affects virtually all newborn infants. Usually, it is an entirely benign process that is resolved at the end of the first week of life without treatment or sequelae. However, in a small percentage of neonates, unconjugated hyperbilirubinemia can pose a neurotoxic risk especially in the presence of aggravating conditions such as a diminished albumin binding capacity and/or affinity, acidosis, displacing drugs and prematurity. Although neuronal cells are considered the main target for unconjugated bilirubin (UCB) toxicity, circulating cells are also affected during neonatal hyperbilirubinemia. Moreover, the UCB ability to cause hemolysis shall further aggravate neonatal jaundice through a vicious circle. In this review, we summarize the most relevant data obtained by our group regarding UCB toxicity and the role of some risk factors for kernicterus. In order to improve the risk assessment of neurotoxicity it is essential to understand the underlying mechanisms of UCB pathophysiology.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. The increased vulnerability of premature infants to unconjugated bilirubin (UCB)-induced brain damage may be due to a proneness of immature nerve cells to UCB-toxic stimulus. Thus, in this study, we evaluated UCB-induced cell death, glutamate release and cytokine production, in astrocytes and neurons cultured for different days, in order to relate the differentiation state with cell vulnerability to UCB. The age-dependent activation of the nuclear factor-kappaB (NF-kappaB), an important transcription factor involved in inflammation, was also investigated. Furthermore, responsiveness of neurons and astrocytes to UCB were compared in order to identify the most susceptible to each induced effect, as an approach to what happens in vivo. The results clearly showed that immature nerve cells are more vulnerable than the most differentiated ones to UCB-induced cell death, glutamate release and tumour necrosis factor (TNF)-alpha secretion. Moreover, astrocytes seem to be more competent cells in releasing glutamate and in producing an inflammatory response when injured by UCB. Activation of NF-kappaB by UCB also presents a cell-age-dependent pattern, and values vary with neural cell type. Again, astrocytes have the highest activation levels, which are correlated with the greater amount of cytokine production observed in these cells. These results contribute to a better knowledge of the mechanisms leading to UCB encephalopathy by elucidation of age- and type-related differences in neural cell responses to UCB.

Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (or=1 microM), for 4 h at 37 degrees C. Intracellular concentrations of reactive oxygen species (ROS) and calcium were determined by dichlorofluorescin and BAPTA fluorescent probes, respectively. Membrane protein and lipid oxidation were evaluated by immunocytochemistry and phosphatidylserine exposure by annexin V binding. Levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as activities of Mg(2+)-ATPase aminophospholipid translocase (flippase) and Na(+),K(+)-ATPase, were also measured. Our results showed that bilirubin induced oxidative stress, due to a rise in lipid (or=10%, P0.05) and protein oxidation (or=20%, P0.01), ROS content (approximately 17%, P0.01), and a decrease in GSH/GSSG ratio (30%, P0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (approximately 10%, P0.05), together with decreased flippase and NA(+),K(+)-ATPase (or=15%, P0.05) activities, events that were accompanied by enhanced intracellular calcium levels ( approximately 20%, P0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.

In hyperbilirubinemic newborns, sepsis is considered a risk factor for kernicterus. Evidence shows that injury to astrocytes triggers cytokine release. We examined the effects of unconjugated bilirubin (UCB) alone, or in combination with LPS, on the release of glutamate and cytokines from astrocytes in conditions inducing less than 10% of cell death. UCB leads to an increase of extracellular glutamate and highly enhances the release of TNF-alpha and IL-1beta, while inhibiting the production of IL-6. LPS potentiates immunostimulatory properties of UCB. These results point out the role of cytokines and provide a basis for the significance of sepsis in UCB encephalopathy.

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0-8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0-7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.

The pathogenesis of cholestasis, bilirubin encephalopathy, and Alzheimer's disease appears to result from accumulation of diverse cytotoxic agents, which in turn may cause apoptotic cell death. In addition, mitochondria has lately been considered as a central executioner of programmed cell death, through the release of caspase activating factors. The aims of this study were to: (a) investigate mitochondrial perturbation during incubation of isolated mitochondria with unconjugated bilirubin (Bb), amyloid beta-peptide (A beta), and glycochenodeoxycholate (GCDC); (b) characterize membrane perturbation in isolated mitochondria induced by each toxic agent, and determine whether the mitochondrial permeabilization is required for cytochrome c redistribution. Mitochondria were isolated from rat liver and brain. Swelling and cytochrome c release were evaluated by spectrophotometry and western blot, respectively. The results showed that Bb as well as A beta and GCDC act directly at the mitochondrial level causing increased organelle volume, permeabilization, as well as cytochrome c release from the intermembrane space in a dose-dependent manner (P0.01). Moreover, cyclosporine A inhibited mitochondrial permeability, particularly after Bb- and GCDC-induced swelling (P0.01). Cytochrome c efflux was invariably prevented by cyclosporine A (P0.05). In conclusion, the results indicate that Bb-, A beta-, and GCDC-induced toxicity, culminating in apoptosis, may result from enhanced mitochondrial permeability, followed by cytochrome c efflux, which can be explained at least in part by the megapore opening.

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Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

Increased levels of unconjugated bilirubin, the end-product of heme catabolism, are detrimental to the central nervous system. To examine the role of apoptosis in bilirubin-induced toxicity and to characterize the biochemical pathway of cell death, we exposed developing rat brain neurons to purified unconjugated bilirubin at concentrations below and above saturation of human serum albumin. Isolated neurons treated with bilirubin showed increased levels of apoptosis. Mitochondrial cytochrome c was extensively released and accumulated in cytosol. Consistent with this observation, caspase-3 was activated and the full-length substrate poly(ADP)ribose polymerase (PARP) degraded, even in the presence of very modestly elevated concentrations of bilirubin. In parallel, all events were prevented in cells preincubated with ursodeoxycholate. Further experiments showed that bilirubin diminished mitochondrial transmembrane potential (DeltaPsi(m)) and increased mitochondrial-associated Bax protein levels, while directly disrupting membrane lipid and protein structure. In conclusion, bilirubin induces mitochondrial depolarization and Bax translocation via physical interaction with membranes, mediating the mitochondrial pathway of apoptosis in neurons exposed to bilirubin. These results provide a novel insight into the mechanism of bilirubin-induced toxicity.

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Background/Aims : Unconjugated bilirubin (UCB) can be neurotoxic in jaundiced neonates and in patients with Crigler–Najjar syndrome. UCB toxicity may culminate in cell death, however, the occurrence of apoptosis has never been investigated. Ursodeoxycholic acid (UDCA) is a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. The aims of this study were to determine whether apoptosis plays a role in neural cell death induced by UCB, and to investigate the ability of UDCA to prevent cell death. Methods : Cultured rat astrocytes were incubated with UCB (17 and 86 μ M) plus albumin (5.7 and 28.7 μ ;M) for 4–22 h. In addition, astrocytes and neurones were treated with either UCB, 50 μ M UDCA, or their combination for 4 h. Cultures were scored for nonviable cells by trypan blue dye exclusion. Apoptosis was assessed by Hoechst staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assay. Results : UCB induced a concentration- and time-dependent decrease in astrocyte viability. Apoptosis was 4- and 7-fold increased after 4 h exposure to 17 and 86 μ ;M UCB, respectively ( P ). UDCA reduced apoptosis to P ). Cholic acid was not protective, and chenodeoxyholic acid aggravated UCB toxicity ( P ). Finally, neurones showed a 1.5-fold greater sensitivity than astrocytes to UCB, while UDCA was still protective. Conclusions : UCB is toxic to both astrocytes and neurones, causing cell death through an apoptotic process. Moreover, UDCA inhibits UCB-induced apoptosis in neural cells and this could not be mimicked by other bile acids.

Unconjugated bilirubin is a neurotoxic pigment that interacts with membrane lipids. In this study we used electron paramagnetic resonance and the spin labels 5-, 7-, 12-, and 16-doxyl-stearic acid (DSA) to evaluate the depth of the hydrocarbon chain at which interaction of bilirubin preferentially occurs. In addition, we used different pH values to determine the molecular species involved. Resealed right-side-out ghosts were incubated (1-60 min) with bilirubin (3.4-42.8 microM) at pH 7.0, 7.4, and 8.0. Alterations of membrane dynamic properties were maximum after 15 min of incubation with 8.6 microM bilirubin at pH 7.4 and were accompanied by a significant release of phospholipids. Interestingly, concentrations of bilirubin up to 42.8 microM and longer incubations resulted in the elution of cholesterol and further increased that of phospholipids while inducing less structural alterations. Variation of the pH values from 8.0 to 7.4 and 7.0, under conditions of maximum perturbation, led to a change from an increased to a diminished polarity sensed by 5-DSA. Conversely, a progressive enhancement in fluidity was reported by 7-DSA, followed by 12- and 16-DSA. These results indicate that bilirubin while enhancing membrane lipid order at C-5 simultaneously has disordering effects at C-7. Furthermore, recovery of membrane dynamics after 15 min of bilirubin exposure along with the release of lipids is compatible with a membrane adaptive response to the insult. In addition, our data provide evidence that uncharged diacid is the species primarily interacting with the membrane as perturbation is favored by acidosis, a condition frequently associated with hyperbilirubinemia in premature and severely ill infants.

The molecular basis of bilirubin toxicity to nerve cell function is still unclear. Since astrocytes are the main transporters of synaptically released glutamate and impaired glutamate uptake results in neuronal death, we investigated the effect of unconjugated bilirubin (UCB) on [3Hlglutamate uptake in cultured rat astrocytes and the role of bilirubin ionization on toxicity. Astrocytes were incubated for 5-15 min with UCB concentrations from 17 to 342 pM and UCB/albumin molar ratios of 0.2-3.0, at pH 7.0, 7.4, and 8.0. Exposure of astrocytes for 15 min to 85.5 1M UCB and 28.5 pM albumin resulted in a 63.1% decrease of glutamate uptake (p < 0.01). Interestingly, the effect demonstrated to be correlated with the UCB/albumin molar ratio (r = -0.986, p < 0.01) and a significant decrease was observed for a UCB/albumin molar ratio as low as 0.8. Inhibition of glutamate transport was also pH-dependent as it occurred at 7.4 (p < 0.05) and 8.0 (p, The authors thank Rosa Santos and Ana Homem for their technical assistance and Lorelia Pascolo and Felicia Cupeili for their comments and help during the experimental approach. We also thank Professor FranÇois Trivin for bis expert advice on astrocyte culture. This work was supported by grants from FundacQ para a Ciência e Tecnologia (PRAXIS/PSAU/C/SAU/127/96), Fondo Studi Fegato, and the Italian Ministry for Research (MURST, Rome).