Molecular events in brain bilirubin toxicity revisited.

The mechanisms involved in bilirubin neurotoxicity are still far from being fully elucidated. Several different events concur to damage mainly the neurons among which inflammation and alteration of the redox state play a major role. An imbalance of cellular calcium homeostasis has been recently described to be associated with toxic concentrations of bilirubin, and this disequilibrium may in turn elicit an inflammatory reaction. The different and age-dependent sensitivity to bilirubin damage must also be considered in describing the dramatic clinical picture of bilirubin-induced neurological damage (BIND) formerly known as kernicterus spectrum disorder (KSD). This review aims to critically address what is known and what is not in the molecular events of bilirubin neurotoxicity to provide hints for a better diagnosis and more successful treatments. Part of these concepts have been presented at the 38th Annual Audrey K. Brown Kernicterus Symposium of Pediatric American Society, Washington DC, May 1, 2023.

Bilirubin 2022: The Past, the Present and the Future.

The present Special Issue (SI) addresses the double-faced Janus behavior of bilirubin [...].

Translational Approach to the Protective Effect of Bilirubin in Diabetic Kidney Disease.

Bilirubin has been regarded as a powerful endogenous antioxidant and anti-inflammatory molecule, able to act on cellular pathways as a hormone. Diabetic kidney disease (DKD) is a common chronic complication of diabetes, and it is the leading cause of end-stage renal disease. Here, we will review the clinical and molecular features of mild hyperbilirubinemia in DKD. The pathogenesis of DKD involves oxidative stress, inflammation, fibrosis, and apoptosis. Serum bilirubin levels are positively correlated with the levels of the antioxidative enzymes as superoxide dismutase, catalase, and glutathione peroxidase, while it is inversely correlated with C-reactive protein, TNF-α, interleukin (IL)-2, IL-6, and IL-10 release in diabetic kidney disease. Bilirubin downregulates NADPH oxidase, reduces the induction of pro-fibrotic factor HIF-1α expression, cleaved caspase-3, and cleaved PARP induction showing lower DNA fragmentation. Recent experimental and clinical studies have demonstrated its effects in the development and progression of renal diseases, pointing out that only very mild elevations of bilirubin concentrations result in real clinical benefits. Future controlled studies are needed to explore the precise role of bilirubin in the pathogenesis of DKD and to understand if the use of serum bilirubin levels as a marker of progression or therapeutic target in DKD is feasible and realistic.

Association of Serum Bilirubin Level with Metabolic Syndrome and Non-Alcoholic Fatty Liver Disease: A Cross-Sectional Study of 1672 Obese Children.

As in adults, obesity also plays a central role in the development of metabolic syndrome (MS) in children. Non-alcoholic fatty liver disease (NAFLD) is considered a manifestation of MS. Not only MS but also NAFLD seem to be inversely associated with serum bilirubin concentrations, an important endogenous tissue protector when only mild elevated. The aim of the study was to evaluate the association between serum bilirubin levels and the prevalence of MS and NAFLD in Italian obese children and adolescents. A retrospective cross-sectional study was performed in 1672 patients aged from 5 to 18 years. Clinical and laboratory parameters were assessed. NAFLD was measured by liver ultrasonography. The study was approved by the Ethical Committee of the Istituto Auxologico Italiano (research project code 1C021_2020, acronym BILOB). MS was present in 24% and fatty liver (FL) in 38% of this population. Bilirubin was not associated with FL and MS as a whole, but it was inversely associated only with selected components of MS, i.e., large WC, high blood pressure and high triglycerides. Our data suggest that bilirubin is not protective against MS and NAFLD in the presence of severe obesity.

Life-Long Hyperbilirubinemia Exposure and Bilirubin Priming Prevent In Vitro Metabolic Damage.

Background: Unconjugated bilirubin (UCB) is more than the final product of heme catabolism. Mildly elevated systemic bilirubin concentrations, such as in Gilbert syndrome (GS), protect against various oxidative stress-mediated and metabolic diseases, including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, cancer, and age-related disease. The Gunn rat is an animal model of hereditary hyperbilirubinemia widely used in assessing the effect of high serum bilirubin concentration in various organs. The present work aims to understand if life-long hyperbilirubinemia and bilirubin-priming might contribute to protection against atherosclerosis and diabetic nephropathy (DN) at the cellular level. Methods: Primary aortic endothelial cells and podocytes obtained from hyperbilirubinemic homozygous jj and normobilirubinemic heterozygous Nj Gunn rats were exposed to Palmitic Acid (PA) and Angiotensin II (Ang II), respectively, and the effects on cell viability and the activation of damage-related metabolic pathways evaluated. Results were validated on immortalized H5V and HK2 cells exposed to damage after UCB pretreatment. Results: In both primary cell models, cells obtained from jj Gunn rats showed as significantly higher than Nj Gunn rats at any dose of the toxic agent. Reduction in CHOP expression and IL-6 release was observed in jj primary aortic endothelial cells exposed to PA compared to Nj cells. The same occurred on H5V pretreated with Unconjugated bilirubin. Upon Ang II treatment, primary podocytes from jj Gunn rats showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction than primary podocytes from Nj Gunn rats. In HK2 cells, the induction by Ang II of HIF-1α and LOXl2 was significantly reduced by UCB pretreatment. Conclusion: Our data suggest that in models of atherosclerosis and DN life-long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to decrease the injury by enhancing thecellular defensive response.

The Extent of Intracellular Accumulation of Bilirubin Determines Its Anti- or Pro-Oxidant Effect.

BACKGROUND: 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.

Bilirubin disrupts calcium homeostasis in neonatal hippocampal neurons: a new pathway of neurotoxicity.

Severe hyperbilirubinemia leads to bilirubin encephalopathy in neonates, causing irreversible neurological sequelae. We investigated the nature of neuronal selective vulnerability to unconjugated bilirubin (UCB) toxicity. The maintenance of intracellular calcium homeostasis is crucial for neuron survival. Calcium release from endoplasmic reticulum (ER) during ER-stress can lead to apoptosis trough the activation of Caspase-12. By live calcium imaging we monitored the generation of calcium signals in dissociated hippocampal neurons and glial cells exposed to increasing UCB concentrations. We showed the ability of UCB to alter intracellular calcium homeostasis, inducing the appearance of repetitive intracellular calcium oscillations. The contribution of intracellular calcium stores and the induction and activation of proteins involved in the apoptotic calcium-dependent signaling were also assessed. Thapsigargin, a specific inhibitor of Sarco/endoplasmic reticulum ATPase (SERCA) pumps, significantly reduced the duration of Ca2+ oscillation associated with UCB exposure indicating that UCB strongly interfered with the reticulum calcium stores. On the contrary, in pure astrocyte cultures, spontaneous Ca2+ transient duration was not altered by UCB. The protein content of GRP78, AT6, CHOP, Calpain and Caspase-12 of neuronal cells treated with UCB for 24 h was at least twofold higher compared to controls. Calcium-dependent Calpain and Caspase-12 induction by UCB were significantly reduced by 50% and 98%, respectively when cells were pretreated with the ER-stress inhibitor 4-PBA. These results show the strong and direct interference of UCB with neuronal intracellular Ca2+ dynamics, suggesting ER Ca2+ stores as a primary target of UCB toxicity with the activation of the apoptotic ER-stress-dependent pathway.

Induction of Mild Hyperbilirubinemia: Hype or Real Therapeutic Opportunity?

Observational epidemiological studies showed that mild hyperbilirubinemia has beneficial effects on the prevention of cardiovascular disease, type 2 diabetes mellitus, and metabolic syndrome. In mammals, bilirubin plays a major role as a potent antioxidant. Uridine 5'-diphospho-glucuronosyl transferase (UGT)1A1 variants coding for bilirubin UDP-glucuronosyl transferase resulting in mild hyperbilirubinemia (as in Gilbert syndrome (GS)) may confer a strong genetic advantage. Strategies to boost bioavailability of bilirubin or to mimic GS represent an attractive approach to prevent many oxidative stress and inflammation-mediated diseases. Even a tiny, micromolar increase in serum bilirubin concentrations substantially decreases the risk of oxidative stress-mediated diseases. There are several possible ways to achieve this, including lifestyle changes, changes in dietary patterns, regular physical activities, or use of chemical drug or of specific plant products either in the form of regular food items or nutraceuticals. Further basic and experimental research is required to fully uncover this promising therapeutic field.

The activation of autophagy protects neurons and astrocytes against bilirubin-induced cytotoxicity.

Unconjugated bilirubin (UCB) neurotoxicity involves oxidative stress, calcium signaling and ER-stress. The same insults can also induce autophagy, a process of "self-eating", with both a pro-survival or a pro-apoptotic role. Our aim was to study the outcome of autophagy activation by UCB in the highly sensitive neuronal SH-SY5Y cells and in the resistant astrocytoma U87 cells. Upon treatment with a toxic dose of UCB, the conversion of LC3-I to LC3-II was detected in both cell lines. Inhibition of autophagy by E64d before UCB treatment increased SH-SY5Y cell mortality and made U87 cells sensitive to UCB. In SH-SY5Y autophagy related genes ATG8 (5 folds), ATG18 (5 folds), p62 (3 folds) and FAM 129A (4.5 folds) were induced 8h after UCB treatment while DDIT4 upregulation (13 folds) started at 4h. mTORC1 inactivation by UCB was confirmed by phosphorylation of 4EBP1. UCB induced LC3-II conversion was completely prevented by pretreating cells with the calcium chelator BAPTA and reduced by 65% using the ER-stress inhibitor 4-PBA. Pretreatment with the PKC inhibitor reduced LC3 mRNA by 70% as compared to cells exposed to UCB alone. Finally, autophagy induction by Trifluoroperazine (TFP) increased the cell viability of rat hippocampal primary neurons upon UCB treatment from 60% to 80%. In SH-SY5Y cells, TFP pretreatment blocked the UCB-induced cleaved caspase-3 protein expression, decreased LDH release from 50% to 23%, reduced the UCB-induction of HO1, CHOP and IL-8 mRNAs by 85%, 70% and 97%. Collectively these data indicate that the activation of autophagy protects neuronal cells from UCB cytotoxicity. The mechanisms of autophagy activation by UCB involves mTOR/ER-stress/PKC/calcium signaling.

Bilirubin-induced ER stress contributes to the inflammatory response and apoptosis in neuronal 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.

Life-long correction of hyperbilirubinemia with a neonatal liver-specific AAV-mediated gene transfer in a lethal mouse model of Crigler-Najjar Syndrome.

Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dl), despite 20-30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.

Bilirubin mediated oxidative stress involves antioxidant response activation via Nrf2 pathway.

Unconjugated bilirubin (UCB) is responsible for neonatal jaundice and high level of free bilirubin (Bf) can lead to kernicterus. Previous studies suggest that oxidative stress is a critical component of UCB-induced neurotoxicity. The Nrf2 pathway is a powerful sensor for cellular redox state and is activated directly by oxidative stress and/or indirectly by stress response protein kinases. Activated Nrf2 translocates to nucleus, binds to Antioxidant Response Element (ARE), and enhances the up-regulation of cytoprotective genes that mediate cell survival. The aim of the present study was to investigate the role of Nrf2 pathway in cell response to bilirubin mediated oxidative stress in the neuroblastoma SH-SY5Y cell line. Cells exposed to a toxic concentration of UCB (140 nM Bf) showed an increased intracellular ROS levels and enhanced nuclear accumulation of Nrf2 protein. UCB stimulated transcriptional induction of ARE-GFP reporter gene and induced mRNA expression of multiple antioxidant response genes as: xCT, Gly1, γGCL-m, γGCL-c, HO-1, NQO1, FTH, ME1, and ATF3. Nrf2 siRNA decreased UCB induced mRNA expression of HO1 (75%), NQO1 (54%), and FTH (40%). The Nrf2-related HO-1 induction was reduced to 60% in cells pre-treated with antioxidant (NAC) or specific signaling pathway inhibitors for PKC, P38α and MEK1/2 (80, 40 and 25%, respectively). In conclusion, we demonstrated that SH-SY5Y cells undergo an adaptive response against UCB-mediated oxidative stress by activation of multiple antioxidant response, in part through Nrf2 pathway.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.

Functional induction of the cystine-glutamate exchanger system Xc(-) activity in SH-SY5Y cells by unconjugated bilirubin.

We have previously reported that exposure of SH-SY5Y neuroblastoma cells to unconjugated bilirubin (UCB) resulted in a marked up-regulation of the mRNA encoding for the Na(+)-independent cystine∶glutamate exchanger System X(c)(-) (SLC7A11 and SLC3A2 genes). In this study we demonstrate that SH-SY5Y cells treated with UCB showed a higher cystine uptake due to a significant and specific increase in the activity of System X(c)(-), without the contribution of the others two cystine transporters (X(AG)(-) and GGT) reported in neurons. The total intracellular glutathione content was 2 folds higher in the cells exposed to bilirubin as compared to controls, suggesting that the internalized cystine is used for gluthathione synthesis. Interestingly, these cells were significantly less sensitive to an oxidative insult induced by hydrogen peroxide. If System X(c)(-) is silenced the protection is lost. In conclusion, these results suggest that bilirubin can modulate the gluthathione levels in neuroblastoma cells through the induction of the System X(c)(-), and this renders the cell less prone to oxidative damage.

A proteomic approach to the bilirubin-induced toxicity in neuronal cells reveals a protective function of DJ-1 protein.

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long-term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB-induced neuronal toxicity, we used the human neuroblastoma cell line SH-SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin-induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells.

BACKGROUND: The deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown. RESULTS: Transcriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented. CONCLUSION: These results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes.

UNLABELLED: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups. IN CONCLUSION: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.

The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1.

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

Cytotoxicity is predicted by unbound and not total bilirubin concentration.

Although it has been suggested that the unbound, free, (B(f)) rather than total (B(T)) bilirubin level correlates with cell toxicity, direct experimental evidence supporting this conclusion is limited. In addition, previous studies never included a direct measurement of B(f), using newer, accurate methods. To test "the free bilirubin hypothesis", in vitro cytotoxicity was assessed in four cell lines exposed to different B(f) concentrations obtained by varying B(T)/Albumin ratio, using serum albumins with different binding affinities, and/or displacing unconjugated bilirubin (UCB) from albumin with a sulphonamide. B(f) was assessed by the modified, minimally diluted peroxidase method. Cytotoxicity varied among cell lines but was invariably related to B(f) and not B(T). Light exposure decreased toxicity parallel to a decrease in B(f). In the absence of albumin, no cytotoxicity was found at a B(f) of 150 nM whereas in the presence of albumin a similar B(f) resulted in a 40% reduction of viability indicating the importance of total cellular uptake of UCB in eliciting toxic effect. In the presence of albumin-bound UCB, bilirubin-induced cytotoxicity in a given cell line is accurately predicted by B(f) irrespective of the source and concentration of albumin, or total bilirubin level.

Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.