DOTTORATO D I R ICERCA IN SC IENZE BIOM OLECO LAR I XX I C IC LO
Settore scientifico-disciplinare: Biochimica (Bio/10)
MECHANISMS INVOLVED IN THE UCB NEUROTOXICITY ON CELLULAR MODELS
Dottorando: Coordinatore del Collegio Docenti:
Pablo José Giraudi Prof. Franco Vittur Università degli Studi di Trieste
Relatore:
Prof. Claudio Tiribelli Università degli Studi di Trieste
Correlatore:
Dott. ssa Cristina Bellarosa Centro Studi Fegato
ANNO ACCADEMICO 2007-2008
A Silvia, Daniela, María y Francisco
que me ayudan a crecer día a día
This study was supported by a fellowship from the Italian Ministry of Foreign
Affairs (MAE) in Rome, Italy. In particular, I wish to thank Dr. Paola
Ranocchia.
Abbreviations I
Summary III
Riassunto V
Chapter 1: General Introduction 1
1. Bilirubin Neurological Diseases 2
1.1. Bilirubin Encephalopathy 2
1.2. Kernicterus 2
1.3. Bilirubin-Induced Neurological Dysfunction 3
2. Chemical-physical characteristics of bilirubin 3
2.1. Structure 3
2.2. Bilirubin ionization and aqueous solubility 4
3. Bilirubin metabolism 5
4. Disorders of bilirubin metabolism 7
4.1. Disorders of bilirubin metabolism characterized by predominantly unconjugated
hyperbilirubinemia 7
4.2. Disorders of bilirubin metabolism characterized by predominantly conjugated
hyperbilirubinemia 9
5. Bilirubin neurotoxicity 10
5.1. The blood - brain barrier 10
5.2. Entry of UCB into brain and protective mechanisms against its neurotoxicity 11
5.3. Molecular basis of UCB neurotoxicity 12
6. Global aims of the thesis 14
7. References 14
Chapter 2: Cellular models for the study of bilirubin toxicity 21
Abstract 22
1. Introduction 23
2. Materials and Methods 24
2.1. Chemicals 24
2.2. Cell culture 24
2.3. UCB solutions and Bf measurements 25
2.6. Immunocytochemistry labelling for Mrp1 27 2.7. RNA isolation, reverse transcription and quantitative PCR 28
2.8. Statistical analysis 29
3. Results 30
3.1. [3H]- Bilirubin uptake by HeLa, 2a1 and SH-SY5Y cells 30 3.2. Bilirubin effect on cell viability in HeLa, 2a1 and SH-SY5Y cells 30
3.3. Localization of Mrp1 in 2a1 and SH-SY5Y cells 31
3.4. Mrp1 and Mdr1 mRNA levels in HeLa and SH-SY5Y cells 32
4. Discussion 33
5 References 34
Chapter 3: Cytotoxicity is predicted by unbound and not total bilirubin concentration 40
Abstract 41
1. Introduction 42
2. Materials and Methods 43
2.1. Chemicals 43
2.2. Cell cultures 43
2.3. Preparations of bilirubin/albumin systems 43
2.4. Bf measurements 44
2.5. Cell viability by MTT reduction 44
2.6. Effect of different albumin preparations on Bf levels and time course of toxicity 44 2.7. Effect of sulfadimethoxine on Bf and cell viability 45
2.8. Effect of light on Bf and cell viability 45
2.9. Statistical analysis 45
3. Results 46
3.1. Effect of different binders on Bf levels 46
3.2. Effect of Bf on cell viability in different cell lines 46 3.3 Effect of sulfadimethoxine on Bf and on viability of SH-SY5Y cells 47
3.4 Effect of Bf on cell viability without albumin 48
3.5 Effect of light on Bf and cell viability 49
4 Discussion 50
5 References 52
Abstract 57
1. Introduction 58
2. Materials and Methods 60
2.1. Chemicals 60
2.2. Cell culture 60
2.3. Treatment of SH-SY5Y cells with Bf (A time course study) 60
2.4. Priming with UCB of SH-SY5Y cells 62
2.5. Determination of intracellular ROS levels and cell proliferation by FACS analysis 62
2.6. Glutathione determinations 63
2.7. Monitoring of cell growth after priming 64
2.8. Response of SH-SY5Y primed cells to a second stress (Bf or H2O2) 64 2.9. Gene expression profile experiments (Microarray analysis) 64
2.10. Real time RT-PCR and Western Blot studies 65
2.11. Statistical analysis 68
3. Results 69
3.1. Sensitivity of SH-SY5Y cells to free bilirubin (Bf) 69
3.2. ROS production in SH-SY5Y primed cells 70
3.3. Intracellular total GSH level in SH-SY5Y primed cells 72
3.4. Growth curve analysis in SH-SY5Y primed cells 74
3.5. Response of SH-SY5Y primed cells to a second stress (Bf or H2O2) 75
3.6. Gene expression profiling induced by UCB 78
3.7. Validation of microarray results by Real time RT-PCR for SLC7A11 gene
and Western Blot for its expression product (xCT) 81
4. Discussion 84
5. References 87
Conclusions and perspectives 93
Acknownledgements 95
List of Publications 96
Abbreviations
ABE Acute bilirubin encephalopathy ABR Auditory brainstem response
APE1/Ref1 Apurinic/apyrimidinic endonuclease 1/redox effector factor
ATP Adenosine triphosphate
BBB Blood Brain Barrier
Bf Free unconjugated bilirubin
BIND Bilirubin induced neurological dysfunction
BSA Bovine serum albumin
BSO Buthionine sulfoximine
BT Total unconjugated bilirubin
CNS Central nervous system
CP Choroid plexus
CSF Cerebrospinal fluid
DEM Diethyl maleate
DMSO Dimethyl sulfoxide
DTNB 5’,5’- dithiolbis-2-nitrobenzoic acid
ER Endoplasmic reticulum
Erg-1 Early growth response 1
FACS Fluorescence activated cell sorting
FCS Fetal calf serum
4F2hc 4F2 cell-surface antigen heavy chain
GSH Reduced glutathione
GSSG Oxidized glutathione
H2DCFDA 2’,7’- dichlorodihydrofluorescein diacetate
HSA Human serum albumin
MDR1 Multidrug resistance protein 1
MEF Mouse embryo fibroblast
MRI Magnetic resonance imaging
MRP1 Multidrug resistance - associated protein 1 MTT Methylthiazoletetrazolium
NADPH Nicotinamide – adenine dinucleotide phosphate
NMDA N- methyl – D – aspartate receptor PBS Phosphate-buffered saline
PMSF Phenylmethylsulphonylfluoride PTEN Phosphatase and tensin homolog
SLC7A11 Solute carrier family 7, (cationic amino acid transporter, y+ system) member 11
SLC3A2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport
UCB Unconjugated bilirubin
xCT Cystine/glutamate transporter
Summary Summary Summary Summary
This doctoral thesis covers three years period (2006-2008) during which I have investigated the bilirubin neurotoxicity in the neuroblastoma SH-SY5Y cell line, a neuronal cell model widely used in the study of the pathogenesis and in the development of new therapeutic compounds for neurodegenerative diseases.
In the first chapter is summarized the current knowledge about bilirubin chemistry and metabolism including disorders of bilirubin metabolism and the neuronal disturbances associated. In addition, the main discoveries in bilirubin toxicity mechanisms are described.
Chapter two describes how we have chosen the cellular model to study the unconjugated bilirubin (UCB) damage. We first compared the bilirubin accumulation and cell viability in two neuronal cell lines (2a1 mouse neuronal progenitor cell line and SH- SY5Y cell line) and one non neuronal cell line (HeLa cells). In addition, we performed studies on cellular localization of Mrp1 (involved in UCB extrusion) and mRNA expression. We observed that SH-SY5Y cells show higher accumulation of bilirubin and lower survival than 2a1 and HeLa cells. SH-SY5Y cells shows a clear localization of Mrp1 at membrane level. Based on these observations we selected the SH-SY5Y cell line as our experimental model, and we characterized this cell line for molecular events linked with bilirubin neurotoxicity.
Chapter three revises original data published by mainly our group, about “the free bilirubin hypothesis”. It has been suggested that cell injury correlates better with free unconjugated bilirubin (Bf) than total unconjugated bilirubin (BT). To directly test this hypothesis we evaluated cell viability in four cell lines (SH-SY5Y, MEF, HeLa and 2a1 cell lines) after incubation with different Bf/BT ratios, obtained by mixing varied UCB concentrations and albumins with different binding affinities (bovine, fetal calf and human); Bf was measured in each solution by the peroxidase method. Our data show that the loss of viability is dependent on the Bf but not on BT although bilirubin sensitivity varied with the different cell line tested. This in vitro study reinforces the proposal that Bf or Bf combined with total serum bilirubin should improve risk assessment for neurotoxicity in both term and premature infants.
Chapter four describes our studies about the biochemical and molecular changes in SH-SY5Y cells exposed to a rather high Bf (140 nM) for 24 hours. Biochemical changes
expression profile were evaluated in the cells which survived after the treatment. Results suggest that the surviving cells become more resistant to a second oxidative exposition (Bf or H2O2) and this was associated with an increases expression of various genes involved both in ER stress response and in the transport system Xc-
(cystine-glutamate exchanger).
This transport system is of great relevance in maintaining the redox homeostasis within the cell, and together with the ER stress genes may contribute to the activation of an adaptative response to bilirubin damage.
Further studies will be necessary to elucidate the molecular mechanisms that confer resistance to bilirubin toxicity; these mechanisms could help understanding the different sensitivity of the cells to bilirubin damage, and why some neuronal cells die (as the Purkinje cells) while others don’t. Furthermore, these studies may achieve to the identification of target proteins useful to develop new drugs: this may be the case of the system Xc-.
Riassunto Riassunto Riassunto Riassunto
Questo lavoro di tesi è il frutto delle ricerche svolte nei tre anni del mio dottorato (2006-2008), durante i quali mi sono occupato dello studio della neurotossicità da bilirubina nella linea cellulare di neuroblastoma umano SH-SY5Y; si tratta di un modello cellulare neuronale ampiamente utilizzato nello studio della patogenesi di malattie neurodegenerative, nonché nello sviluppo di composti neuroprotettivi.
Nel primo capitolo si trovano riassunte le conoscenze attuali riguardanti la chimica della bilirubina, il suo metabolismo ed eventuali disordini ed i disturbi neuronali associati ad essa; inoltre, sono descritte le principali scoperte sui suoi meccanismi di tossicità.
Nel secondo capitolo viene descritta come è stata effettuata la scelta di un modello cellulare adeguato allo studio del danno da bilirubina non coniugata (UCB). A questo scopo sono stati confrontati l’accumulo in bilirubina triziata e la vitalità cellulare dopo un trattamento con bilirubina libera, in due linee cellulari neuronali (progenitori neuronali di striato di topo -cellule 2a1- , neuroblastoma umano -cellule SH-SY5Y-) ed in una linea cellulare non neuronale (cellule HeLa). Oltre a ciò, sono stati eseguiti alcuni studi sulla localizzazione del trasportatore Mrp1 (coinvolto nell’estrusione di UCB), e sull’espressione dei geni Mrp1 ed Mdr1 (il cui prodotto proteico è un possibile trasportatore di bilirubina). Abbiamo osservato che le cellule SH-SY5Y presentano un accumulo di bilirubina più elevato ed una più bassa sopravvivenza rispetto alle cellule 2a1 ed HeLa, sebbene nelle cellule SH-SY5Y la localizzazione di Mrp1 risulti essere a livello di membrana plasmatica. Basandoci su queste osservazioni abbiamo scelto di lavorare con il modello cellulare già noto SH-SY5Y, e ci siamo occupati di caratterizzarlo per la neurotossicità da bilirubina.
Nel terzo capitolo vengono presentati dati sperimentali pubblicati dal nostro gruppo a supporto dell’ “ipotesi della bilirubina libera”, la quale postula che il danno cellulare da bilirubina correli in modo migliore con la concentrazione di bilirubina libera (Bf) piuttosto che con quella di bilirubina totale (BT). Al fine di testare quest’ipotesi abbiamo valutato la vitalità in quattro diverse linee cellulari (SH-SY5Y, MEF, HeLa e 2a1) dopo aver incubato le cellule in soluzioni con un diverso rapporto Bf/BT. Tali soluzioni sono state ottenute sciogliendo diverse quantità di UCB in terreno con diversi tipi di albumina (bovina, umana e di siero fetale bovino); questi binders possiedono differenti affinità per la bilirubina. La Bf è stata determinata in ciascuna soluzione utilizzando il metodo della perossidasi. I dati
ottenuti suggeriscono che, sebbene la sensibilità alla bilirubina vari nelle diverse linee cellulari, la riduzione in vitalità dipenda dalla Bf e non dalla BT. Quindi, questi studi in vitro costituiscono un’evidenza in più a favore della teoria della bilirubina libera, e sostengono la necessità di valutare il rischio di Kernittero mediante la misura della Bf serica e non solo della bilirubina totale.
Nel quarto capitolo si descrivono le modificazioni a livello biochimico e molecolare nella linea cellulare SH-SY5Y dovute ad un trattamento di 24 ore in presenza di un’elevata concentrazione di bilirubina libera. Nelle cellule sopravvissute al trattamento abbiamo valutato diversi parametri biochimici tra cui vitalità e proliferazione cellulare ed ambiente redox cellulare (contenuto di ROS e GSH), nonché il pattern di espressione genica indotto dalla bilirubina. I risultati ottenuti suggeriscono che le cellule SH-SY5Y sopravvissute siano più resistenti all’esposizione ad un secondo stress ossidativo (Bf o H2O2), inoltre queste cellule mostrano un’aumentata espressione di diversi geni coinvolti nella risposta allo stress di reticolo endoplasmatico e dei geni i cui prodotti proteici fanno parte del sistema di trasporto Xc- (antiporto cistina-glutammato). Questo sistema di trasporto è estremamente importante nel mantenimento dell’omeostasi redox cellulare, ed insieme ai geni dello stress di ER potrebbe contribuire all’attivazione di una risposta adattativa al danno da bilirubina.
Ulteriori studi che ci consentano di comprendere i meccanismi molecolari che conferiscono resistenza alla neurotossicità da bilirubina potrebbero aiutarci a capire la differenza di sensibilità dei diversi tipi di cellule alla bilirubina stessa, ed il motivo per cui alcune cellule neuronali muoiano (come ad esempio le cellule di Purkinje) mentre altre no.
Inoltre questi studi possono portarci all’identificazione di target proteici utili allo sviluppo di nuovi farmaci, quale può essere ad esempio il caso del trasportatore Xc-.
Chapter 1
General Introduction
1. Bilirubin Neurological Disease
Although originally a pathologic diagnosis characterized by bilirubin staining of the brainstem nuclei and cerebellum, the term “kernicterus” has come to be used interchangeably with both the acute and chronic findings of bilirubin encephalopathy.
Bilirubin encephalopathy describes the clinical central nervous system findings caused by bilirubin toxicity to the basal ganglia and various brainstem nuclei. To avoid confusion and encourage greater consistency in the literature, the Committee for Quality Improvemment and Subcommittee on Hyperbilirubinemia of the American Academy of Pediatrics (AAP) recommends that in infants the term “acute bilirubin encephalopathy” be used to describe the acute manifestations of bilirubin toxicity seen in the first weeks after birth and that the term “kernicterus” be reserved for the chronic and permanent clinical sequelae of bilirubin toxicity (1).
1.1 Bilirubin Encephalopathy
The classical clinical expression of acute bilirubin encephalopathy (ABE) consists of decreased feeding, lethargy, variable abnormal tone (hypotonia or hypertonia), highpitched cry, retrocollis and opisthotonus, setting sun sign, fever, seizures, and death. Laboratory evidence ranges from increased abnormal auditory brainstem respons (ABR) interwave intervals I–III and I–V and decreased amplitude waves III and V to absent ABRs, and magnetic resonance imaging (MRI) shows acute abnormalities in the globus pallidus and subthalamic nucleus. Abnormal ABRs may improve or normalize with exchange transfusion (2) (3).
1.2 Kernicterus
Kernicterus is a severe and life-threatening condition with an incidence of less than 1 of 30,000 jaundiced neonates, described for the first time by Jaques F. É. Hervieux in 1847
(4). Kernicterus causes selective yellow staining in the basal ganglia, especially the globus pallidus and subthalamic nucleus. Brainstem nuclei, especially the auditory (cochlear nucleus, inferior colliculus, superior olivary complex), oculomotor and vestibular nuclei are especially vulnerable. Other susceptible areas are the cerebellum, especially Purkinje cells, and the hippocampus especially the CA2 sector. The basal ganglia lesions are clinically correlated with the movement disorders of dystonia and athetosis. Abnormalities of the auditory brainstem nuclei are associated with deafness, hearing loss, and a recently
described entity known as auditory neuropathy (AN), also known as auditory dys- synchrony (AD). Abnormalities of the brainstem oculomotor nuclei are associated with strabismus and gaze palsies, especially paresis of upgaze (5).
The basal ganglia can be imaged with MRI, the signature of which is bilateral damage of the globus pallidus. The subthalamic nucleus can sometimes be seen and is characteristically affected.
In conclusion, kernicterus is a complex clinical and neuropathological syndrome where 70% of children with kernicterus die within seven days, while the 30% survivors usually suffer the irreversible described neurological sequelae. The clinical expression of bilirubin neurotoxicity varies with location, severity, and time of assessment, and is influenced by factors including the amount, duration and developmental age of exposure to excessive free bilirubin. Although total serum bilirubin is an important risk factor, kernicterus cannot be defined based on total serum bilirubin alone. Wennberg suggested that measurement of free bilirubin in newborns with hyperbilirubinemia will improve risk assessment for nurotoxicity (6). Finally, Shapiro suggest that kernicterus may be defined for study purposes in term and near-term infants with total bilirubin 20 mg/dl using abnormal muscle tone on neurological examination, auditory neurophysiological testing ABR and MRI (5).
1.3 Bilirubin-Induced Neurological Dysfunction
The term Bilirubin-Induced Neurological Dysfunction (BIND) is described by AAP as a scoring scale to evaluate the severity of acute bilirubin encephalopathy in regards to the need of treatment of infants. The BIND score has not yet been validated but it will comprise a spectrum of permanent sequelae seen (often in the subtle, non-classical sequelae) due to less severe hyperbilirubinemia (7).
2. Chemical – physical characteristics of bilirubin
2.1 Structure
Unconjugated bilirubin (UCB) is a nearly symmetrical tetrapyrrole, consisting of two rigid, planar dipyrrole units, joined by a methylene bridge at carbon 10. The structure thus resembles a two bladed propeller, in which the blades could theoretically be joined at different angles and each blade could rotate about its bond to the methylene bridge (8). In
the preferred “ridge-tile” conformation, the two dipirrinones are synperiplanar, as in a partially opened book, and the angle (θ) between the two planes is about 95°.
The rigid biplanar structure of bilirubin IXα the most naturally occurring isomer
(9)(Figure 1.1), with its internal hydrogen bonds, was first demonstrated in the crystalline state by X-ray diffraction (10), but is also the preferred conformation in solutions of UCB in water, alcohols, and chloroform (11).
2.2 Bilirubin ionization and aqueous solubility
At physiological pH values in plasma (7.4), tissues (7.6) and bile (6.0 to 8.0) there is significant ionization of the –COOH groups of the natural IXα isomer of UCB (12), so that, in addition to the diacid (H2B), a proportion of UCB is present as monoanion (HB-) and dianion (B2-). The pK’a values of the –COOH groups on the two carboxymethyl (propionyl) sidecahins determine the proportions of the free UCB species at any given pH.
The calculated proportions of the individual UCB species in aqueous solution at pH values from 6.0 to 8.0, using the partition-derived experimental pK’a values (12), show that H2B is the dominant species and HB- the dominant anion over this physiological pH range. The bilirubin dianion B2- is a significant fraction only at pH 7.2 (Figure 1.2).
Figure 1.1. The structure of bilirubin IXα-Z,Z, diacid (H2B), which consist of two slightly asymmetrical, rigid, planar dipyrrinone chromophores, connected by a central –CH2- bridge. (Adapted from Pu et al.
Tetrahedron 47: 6163-6170)
The low aqueous solubility of UCB diacid is probably due to its many hydrophobic groups and the internal hydrogen bonding of all its polar groups precluding their interaction with water (13;14). Experimental solubility near to neutral pH, determined by chloroform-to-water partition (12), indicate that the maximum aqueous solubility of H2B at 25°C and ionic strength 0.15, is about 70 nM. Bilirubin solubility increases with increasing pH due to successive ionization of H2B to HB- and B2-.
3. Bilirubin Metabolism
Bilirubin is the oxidative product of the protoporphyrin portion of the heme group present in haemoglobin, myoglobin, and some enzymes. An adult healthy person produces 250-400 mg of bilirubin per day (15). More than 80% of the bilirubin produced in the human body derives from heme catabolism liberated from senescent red cells, 15-20%
derives from the turnover of myoglobin, cytochromes and other hemoproteins, and less than 3% derives from destruction of immature red blood cells in the bone marrow (15;16).
Heme degradation is performed by the reticuloendothelial enzyme heme oxigenase, which is particularly abundant in spleen and liver Kupffer cells, the principal sites of red cell breakdown. This enzyme directs stereospecific cleavage of the heme ring, freeing the iron ion and forming a tetrapyrrolic chain with the final formation of biliverdin and carbon monoxide (17). This reaction requires a reducing agent, such as, nicotinamide-adenine dinucleotide phosphate (NADPH) and three molecules of oxygen. Following its synthesis biliverdin is converted to bilirubin by the cytosolic enzyme biliverdin reductase, in the presence of NADPH (Figure 1.3).
Figure 1.2. Proportions of unbound species of unconjugated bilirubin at pH 6.0 to 8.0, derived from partitions of UCB from chloroform into buffered NaCI at ionic strengh 0.15. Adapted from Hahm et ak, J. Lipid Rrs. 33: 1123-1137
Once released in the blood and due to its poor aqueous solubility, bilirubin is tightly but reversibly binds to serum albumin. Albumin binding keeps bilirubin in solution and transports the pigment to different organs and to the liver in particular. Albumin binds almost the total bilirubin and less than 0.1% of the pigment is unbound to albumin. This small unbound fraction of bilirubin (Bf) is thought to be responsible for its biological effects (18-20).
Despite high-affinity binding to albumin, bilirubin is rapidly transferred from plasma into the liver. At the sinusoidal surface of the hepatocytes, bilirubin dissociates from albumin and is internalized, though mechanism not yet fully clarified. Although it is known that bilirubin uptake is saturable, indicating a carrier-mediated process, the molecules responsible for this transport are still underdetermined (21;22). Once within the aqueous environment of the hepatocyte, bilirubin is again bound to a group of proteins, mainly to glutathione-S-transferases (23;24). These cytosolic proteins are of importance in diminishing reflux of unconjugated and conjugated bilirubin back into the plasma.
Figure 1.3. Bilirubin metabolism. Bilirubin derives from heme metabolism by heme- oxigenase and biliverdin reductase.
Inside of the endoplasmic reticulum, bilirubin is conjugated with either one or two molecules of glucuronic acid by the enzime UDP-glucuronosyltransferase 1A1 (UGT).
These mono- and di- glucuronides display high polarity, which renders then water soluble and unable to diffuse across membranes, and allows their secretion into the bile canaliculus by the membrane transporter multidrug resistance protein 2 (MRP2 or ABCC2).
Conjugated bilirubin excreted in bile passes through the small intestine without significant absorption. In the colon, it is both deconjugated, presumably by the bacterial β- glucuronidase, and degraded by other bacterial enzymes to a large family of reduction- oxidation products, collectively known as urobilinoids, which are mostly excreted by feces.
4 Disorders of bilirubin metabolism
Hepatic transport of bilirubin involves four distinct but probably interrelated stages:
a) uptake from the circulation; b) intracellular binding or storage: c) conjugation, largely with glucuronic acid; and d) biliary excretion. Abnormalities in any of these processes may result in hyperbilirubinemia. In several inheritable disorders, the transfer of bilirubin from blood to bile is disrupted at a specific step. Study of these disorders has permitted better understanding of bilirubin metabolism in health and disease. Each disorder is characterized by varied degrees of hyperbilirubinemia of the unconjugated or conjugated type (25).
4.1 Disorders of bilirubin metabolism characterized by predominantly unconjugated hiperbilirubinemia
Neonatal hyperbilirubinemia
In general, neonates are not jaundiced at the moment of birth because of the ability of the placenta to clear bilirubin from the fetal circulation. At birth, this placental protection is suddenly lost, just when an acute increase in production of unconjugated bilirubin occurs, due to the shorter red blood cells life span of newborns (70-90 vs. 120 days in adults), especially in prematures. In addition, the newborn has to use its own immature mechanisms for hepatic uptake, conjugation and biliary secretion of bilirubin. All these reasons together explain the significant retention of UCB occurring in almost all healthy term neonates (9;26;27). Such retention is further enhanced by the absence of intestinal bacterial flora in the newborn infant, leading to more unmetabolized UCB available for intestinal absorption, thus increasing the enterohepatic circulation of UCB (28). As a result, about half of all neonates become clinically jaundiced during the first days of life, with
moderate to elevated serum UCB levels that returned to normal within 7 to 10 days with not treatment requirement (27;29). This condition is known as “physiologic jaundice”. This neonatal jaundice reflects the transition from intrauterine to extrauterine bilirubin metabolism; it is considered benign and is linked to normal development.
However, in some newborns plasma UCB levels can increase dramatically and expose the baby to the risk of kernicterus. Although plasma bilirubin levels of 20 mg/dL or higher are considered dangerous, bilirubin encephalopathy may occur at lower concentrations. Phototherapy and exchange transfusions are two therapeutic options in the neonate, which have significantly reduced the prevalence of bilirubin encephalopathy (30).
Crigler-Najjar Syndrome, Type I and II
Crigler-Najjar syndrome tipe I is a rare genetic disorder (one in 1.106 newborns) described by Crigler and Najjar in 1952 (31) and is characterized by sever unconjugated hyperbilirubinemia due to the absence of the UGT enzyme. Serum bilirubin is virtually all unconjugated and the levels typically range from 15 to 45 mg/dL and, if untreated by phototherapy, may result in kernicterus with permanent neurological damage, or death. At present, the only cure is orthotropic liver transplantation (OLT), which permanently corrects the metabolic defect (32).
Crigler-Najjar syndrome, type II is differentiated from type I by reduction of serum bilirubin levels on treatment with phenobarbital or other agents which induce hepatic microsomal enzymes (33;34). Phenobarbital functions via a phenobarbital-responsive enhancer module which stimulates the UGT 1A1 gene to induce production of the bilirubin conjugating enzyme (35).
Many different mutations in the UGT gene have been identified, which may cause both types I and II Crigler Najjar syndrome. Most of these occur in the coding region of the gene. Mutations leading to type I syndrome are mainly missense mutations, nonsense mutations, mutations leading to a premature stop codon, mutations leading to frame shifts and splice site mutations, all leading to no enzyme-proteins, truncated enzymes or inactive full-length enzyme proteins. Missense mutations found in association with type II Crigler- Najjar syndrome result in partial enzyme activity (36).
An animal model of Crigler-Najjar Type I
Mutant Wistar rats with non-hemolytic unconjugated hyperbilirubinemia were described by Gunn in 1938. Homozygous Gunn rats lack UGT activity and are prototypes of Crigler-Najjar syndrome, type I. The molecular basis of UGT1A1 deficiency in this strain is the deletion of a guanosine base in the common region exon 4. Gunn rats have 3 to 20 mg/dL of serum bilirubin, all of which is unconjugated. Heterozygous Gunn rats are anicteric. Homozygous Gunn rats develop cytoplasmic neuronal changes on the third day of life; and degeneration of Purkinje cells and other neuronal cells is evident by 2 weeks.
The brain of a healthy Gunn rat does not have yellow staining.
Gilbert Syndrome
It is a common, benign condition frequently encountered (3-5%) in healthy adults.
Gilbert syndrome is suspected when routine tests show an increased total serum bilirubin (1-5 mg/dL), almost completely in the unconjugated moiety, without signs of increased hemolysis and with normal routine liver function test and hepatic histology (25). The most common genetic polymorphism encountered in association with Gilbert’s syndrome is that of an additional TA insertion in the TATAA box of the UGT 1A1 promoter, identified as UGT1A1*28, although not all individuals homozygous for the promoter mutation manifest clinical signs of this condition (36).
4.2 Disorders of bilirubin metabolism characterized by predominantly conjugated hyperbilirubinemia
Dubin-Johnson Syndrome and Rotor’s syndrome
Dubin-Johnson syndrome is characterized by chronic intermittent conjugated hyperbilirubinemia and black pigmentation of the liver without other abnormalities of clinico-chemical test for liver dysfunction (37;38). The cause of Dubin-Johnson syndrome is a nonsense mutation of the coding region of the gene for MRP2 (ABCC2), the canalicular membrane transporter that normally extrudes a vast number of metabolites into bile, including conjugated bilirubin. Bilirubin glucuronides reflux back into blood creating a typical pattern of conjugated hyperbilirubinemia and are excrete by the kidneys causing bilirubinuria.
Rotor’s syndrome is characterized by accumulation of conjugated bilirubin also in presence of normal liver function test. In contrast to Dubin-Johnson syndrome, there is no increased pigmentation of the liver. Its molecular basis is unknown.
Onset is typically in adults although it may rarely become manifest in infancy as severe cholestasis. Both syndromes have an excellent prognosis (39).
5. Bilirubin neurotoxicity
Recent increases in the prevalence of bilirubin encephalopathy and its occasional occurrence at plasma bilirubin levels below therapeutic guidelines (40) have revived interest in understanding the mechanisms of UCB-induced neurotoxicity (41). Neurotoxicity is determined primarily by the free bilirubin (Bf), the concentration of the unbound free fraction of UCB in plasma. Below the aqueous solubility limit of 70 nM at pH 7·4, unbound UCB is exclusively in the form of monomers and small oligomers (12). When Bf is modestly above saturation, UCB diacid forms soluble oligomers and charge-stabilized, metastable microaggregates (42). It is only at higher Bf values (roughly approximately 1 µM) that insoluble precipitates of UCB diacid form (12), which have long been believed to cause frank kernicterus (43).
5.1 The blood-brain barrier
Except for the circumventricular region of the brain, penetration of drugs and other compounds into the cerebrospinal fluid (CSF) and brain parenchyma is limited by two barriers: the choroid plexus (CP, the blood–CSF barrier) and the brain capillary endothelium (the blood–brain barrier, BBB). The endothelial cells of the BBB have tight junctions and no fenestrae, so they severely restrict both paracellular and transcellular diffusion of many toxic compounds to the adjacent neurons and astrocytes. In the CP, the endothelial cells lack tight junctions and are fenestrated, allowing some access of plasma proteins and their bound ligands to the CSF.
5.2 Entry of UCB into brain and protective mechanisms against its neurotoxicity
In contrast to other reservoirs for bilirubin binding, the brain is unique by having a BBB that reduced the velocity at which equilibrium between plasma and brain is achieved.
If the BBB is disrupted, the complex bilirubin-albumin rapidly moves into the extracellular space of brain, and bilirubin will produce immediate global neurotoxicity (44;45). When the BBB is intact, the rate of bilirubin uptake by brain is determined by: a) the Bf concentration and Bf uptake presumably mediated by OAPTs/Oatps transporters (46); b) the permeability and surface area of the capillary endothelium; c) the transit time through the capillary bed; d) the dissociation rate of bilirubin/albumin, K-1; and e) the blood flow (47). Bilirubin uptake may be increase by alterations in BBB permeability to Bf or albumin (e.g.
hyperosmolality, sever asphyxia), prolonged transit time (e.g. increased venous pressure), an increase in blood flow (e.g. hypercarbia), or an increase in the dissociation rate (e.g.
altered albumin binding in sick infants).
Net transport of bilirubin across the BBB may also be influenced by the energy dependent multidrug resistant transporters, MDR1 and MRP1. MRD1, or P-glycoprotein, is one of several transporters involved in cellular efflux of xenobiotics, and is expressed in capillary endothelial cells of the BBB, astrocytes, and the choroids plexus (46). In a study by Watchko (48), brain uptake of bilirubin in Mdr1a-/- knockout mice infused with high concentrations of bilirubin was twice that of controls ( Mdr1+/+). Inhibition of P- glycoprotein augments bilirubin-induced apoptosis in a human neuroblastoma line (49) and increases bilirubin content in brains of young adult rats (50).
Figure 1.4. Schematic representation of the organization of the brain and its barriers, including the distribution and, where known, polarity (arrows) of the MDR1/Mdr1 and MRP1/Mrp1 transporters in the various cell types. (Adapted from Ostrow J.D. et al. European Journal of Clinical Investigation 33: 988-997).
The multidrug-resistance–associated protein, MRP1, performs similar functions.
MRP1 is highly expressed in choroid plexus epithelium, astrocytes, (rat) neurons, and placenta trophoblast but has minimal expression in capillary endothelium in whole brain
(51). It is upregulated in cultured cells that are exposed to unconjugated bilirubin and mediates ATP-dependent cellular export of bilirubin (52). Inhibition of cultured astrocytes with a non-specific MRPs inhibitor increases apoptosis induced by low concentrations of bilirubin (53). More relevant is the observation that MRP1 is able to bind UCB with the highest affinity so far reported (10 nM) (54) and MEF (mouse embryonic fibroblast) cells from Mrp1 KO rats show a higher UCB toxicity (55). Recently Corich demonstrate in neuroblastoma SH-SY5Y cells, by small interfering RNA of MRP1 and MDR1 that, at clinically-relevant Bf levels, protection from UCB cytotoxicity was correlated with the level of expression of MRP1 but not MDR1 (56).
Other potential cellular defense mechanisms include: a) mitochondrial bilirubin oxidation that has been demonstrated in guinea pig and rat brain (57;58), but its distribution in the central nervous system (CNS) and role in jaundiced animals are unknown; b) binding of bilirubin to cytosolic proteins (e.g. glutathione-S-transferases); and c) anti- apoptosis factors as neuronal apoptosis inhibitors proteins (NAIPs) that inhibit, caspases 3, 7 and other proteins in the apoptotic pathways (59;60).
5.3 Molecular basis of UCB neurotoxicity
The toxic effect of bilirubin in the brain of neonates has been observed since ancient times. The study of morphological changes associated with hyperbilirubinemia has been ongoing for decades, and yet the most basic question of the cytotoxicity of bilirubin and pathogenesis of observed associated lesions remains unresolved.
Bilirubin exhibits a wide range of toxic effects in cell culture systems and in cell homogenates. Bilirubin inhibits DNA synthesis in a mouse neuroblastoma cell line (61), and uncouples oxidative phosphorylation and inhibits adenosine triphosphatase (ATPase) activity of brain mitochondria (62). In mutant Gunn rats with congenital nonhemolytic hyperbilirubinemia, bilirubin inhibited RNA and protein synthesis, and the carbohydrate metabolism in brain (63). In developing rat brain neurons, UCB permeabilizes mitochondrial membranes (64), (65)
, leading to mitochondrial swelling and the release of cytochrome c into the cytosol (66). This triggers activation of caspase-3 and translocation of bax, resulting in cell death by apoptosis via the mitochondrial pathway (67;68). Interestingly,
the level of cell death by apoptosis is similar to that of necrosis in cultured astrocytes exposed to UCB, suggesting that UCB-induced cell death is not linked to a single pathway
(69-71)
. In a recent study, decreased oligodendroglial cell viability and increased apoptosis following exposure to UCB were also observed (72). In addition to the role of mitochondria in mediating apoptotic cell death initiated by UCB, activation of caspase 8, an initiator of apoptosis, suggests that the cell-surface death receptor pathway might be amplified by UCB-induced enlargement of the rough endoplasmic reticulum, which has direct effects on intracellular Ca2+ (73;74). Indeed, in a cell-free system, bilirubin inhibited Ca2+-activated, phospholipid-dependent, protein kinase (PKC) activity and 3’,5’-cyclic adenosine monophosphate (cAMP)-dependent protein kinase activity (75) may be relevant in the mechanisms of its toxicity. Recently, Cesaratto and Calligaris demonstrated using HeLa and mouse embryonic fibroblasts that bilirubin modulates a signalling pathway involving APE1/Ref-1 (a master redox regulator in eukaryotic cells), Egr-1 and PTEN (76). Collectively, these observations point to the disruption of several vital functions by UCB, rather than a single cell-death pathway.
On the other hand, the effects of UCB on neuronal excitability has been the subject of several studies. UCB has significant inhibitory effects on the nervous system, end even short-term exposure to UCB can inhibit long-term exposure potentiation of synaptic transmission in the hippocampus (77), which is an important function affecting learning and memory. This inhibition is in accordance with previous observations showing that UCB inhibits protein kinase C activity in cultured neurons (78). This activity is essential to maintain the synaptic activity that results from an influx of Ca2+ through a high conductance cation channel in the plasma membrane of the post-synaptic neuron. The opening of this channel is triggered by depolarization of the membrane, as well as the binding of glutamate to N-methyl-D-aspartate (NMDA class 1) receptors in the membrane
(79). The mechanism by which UCB disrupts cell homeostasis might also be related to the direct interaction of UCB with nerve cell membranes, which increase oxidative damage and membrane permeability and decreases lipid and protein order (80).
6. Global aims of the thesis
The main goal of the present work is to further contribute to a better knowledge of the molecular mechanisms underlying neonatal hyperbilirubinemia neurotoxicity particularly in the early stage. This thesis has three specific aims:
1) To clarify, in vitro system if bilirubin cytotoxicity correlates with free bilirubin or total bilirubin concentration,
2) To study biochemicals and genetic changes caused by bilirubin causes in the in vitro system,
3) To identify potential drug targets to prevent bilirubin neurological damage.
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Chapter 2
Cellular models for the study of bilirubin toxicity
Cristina Bellarosa, Pablo Giraudi Pablo Giraudi Pablo Giraudi, Sebastian Calligaris, J. Donald Pablo Giraudi Ostrow and Claudio Tiribelli
Parts of this study was presented at the Annual Meeting of the
Pediatric Academic Societies in Toronto, 2007
Abstract
Unconjugated bilirubin (UCB) is neurotoxic and high UCB serum levels in newborns can produce brain damage (kernicterus). Neurotoxicity correlates best with the plasma concentration of the unbound (free) bilirubin (Bf) than total unconjugated bilirubin (UCB).
Previous data demonstrated that different cell lines sustain different extent of damage by bilirubin under the same experimental conditions, but in these works the Bf was not evaluated. In this study we selected an appropriate cellular model to study the bilirubin damage. To this purpose, we compared the UCB cellular uptake and cell viability after 4h of treatment with different Bf (10, 40 and 80 nM) on two neuronal (SH-SY5Y and 2a1 cells) and one non-neuronal (HeLa cells) cell lines. In addition, we studied: the localization of Mrp1, involved in UCB extrusion, in SH-SY5Y and 2a1 cells and the mRNA expression of Mrp 1 and Mdr1, a putative UCB transporter, in HeLa and SH-SY5Y cells. Results show that SH-SY5Y cells are the most sensitive to Bf cytotoxicity and this correlate with a higher capacity of UCB uptake. SH-SY5Y cells present Mrp1 mainly localized at membrane level. These observations point to the well-known SH-SY5Y cell line as a good model to study the intracellular mechanisms of UCB neurotoxicity.
1. Introduction
Unconjugated bilirubin (UCB) has been found to be toxic to many cell examined in vitro, including fibroblasts (1), (2), hepatocytes, erytrocytes, leukcocytes (3), HeLa (4), (5), primary cultures of astrocytes and neurons (6), mouse embryo fibroblast (7), NT2 cells (8), 8402 human cells (9) and neuronal cell lines (10), (11), (12)
. Since UCB can diffuse into any cell (13), (14)
and is a potent antioxidant at low concentrations but toxic at high concentrations (15), all cells must maintain the intracellular concentration of UCB below toxic concentrations. This is regulated by consumption (conjugation and oxidation), and export of UCB (16). Since, unlike hepatocytes, most cells possess low conjugation activity or do not possess it, they have to oxidize or export UCB to prevent its intracellular accumulation.
Several studies in vitro have showed that MRP1/Mrp1 (multidrug resistance- associated protein 1) transports the UCB (17), (18) with an affinity (Km = 10 nM) (19) that is 10 times more than other substrates and protects the cells against its accumulation and toxicity
(20), (21), (22)
. UCB is considered a potential substrate also for Mdr1 or P-glycoprotain (multidrug resistance protein 1), an ATP-dependent plasma membrane efflux pump expressed in brain capillary endothelial cells and astrocytes (23), (24).
Serveral in vitro studies on bilirubin toxicity have demonstrated that the UCB damage is different, depending on the cell type. We have recently demonstrated (25) (See chapter 3) that is necessary to measure free bilirubin (“Bf the real damaging player”) because it would facilitate interpretation and comparison of studies conducted in different laboratories under different conditions. These shortcoming greatly contributed that the molecular mechanisms of bilirubin toxicity are still not fully understood.
As regards of these molecular mechanisms, various observations (as already described in “General Introduction”) suggests that the damage is initiated at the level of membranes (plasmatic, mitochondrial, and ER) with resultant perturbations of membrane permeability and function (26;27), (28)
. These perturbations will contribute to the genesis of neuronal excitotoxicity (29), (30)
, mithocondrial energy failure (31), (32), (33), (34), (35)
and increased intracellular Ca2+ concentration (36). Collectively, these three phenomena and downstream events trigger cell death by both apoptosis and necrosis (37),(38;39), (40)
.
In our laboratory three different cell lines were available: one was the HeLa cell line (human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18) and two neuronal cell lines: 2a1 (neuronal cell line from
mouse striatum) and SH-SY5Y cell (a third generation neuroblastoma, cloned from SK-N- SH isolated from a woman’s metastatic bone tumor). Since we were very interested in selecting the more appropriate cellular model to study the bilirubin damage, we will describe the characterization of the selected model.
To choose the model we first compared the cellular uptake and accumulation of [3H]-bilirubin between the three cell lines. We then studied the correlation between the cellular accumulation of UCB and changes in cell viability, and we finally investigated the putative transporters involved in the extrusion of UCB out of the cell by analyzing: a) localization of Mrp1 in 2a1 and SH-SY5Y cells, b) quantification of relative expression of Mrp1 and Mdr1 in HeLa and SH-SY5Y cells.
2. Materials and Methods 2.1 Chemicals
Unconjugated bilirubin (UCB)(Sigma Chemical Co, St. Louis MO), was purified as described by Ostrow & Mukerjee (41). [3H]UCB (29.3 mCi/mmol) was biosynthetically labeled in vivo and then highly purified from the bilirubin conjugates in bile as described
(42). Dulbecco’s Phosphate Buffered saline (PBS), Dulbecco’s modified Eagle’s medium high glucose (DMEM/high glucose), streptomycin and penicillin were purchased from Euroclone, Milan (Italy). Ham’s Nutrient Mixture F12 (F12), Eagle’s Minimum Essential Medium (EMEM), nonessential amino acid solution (MEM), fatty acid free bovine serum albumin fraction V (BSA), 3(4,5-dimethiltiazolil-2)-2,5 diphenyl tetrazolium (MTT), dimethy sulfoxide (DMSO), horseradish peroxidase (HRP type I) and Tri Reagent were purchased from Sigma Chemical Co.-Aldrich, Milan (Italy). Fetal calf serum (FCS) and GlutaMAXTM, obtained from Invitrogen (Carlsbad, CA) contained 24 g/L albumin.
Chloroform (HPLC grade) was obtained from Carlo Erba, Milan (Italy). iScriptTM cDNA Synthesis kit, iQTM SYBR Green Supermix were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Polyclonal monospecific anti-Mrp1 A-23 antibody was developed in our laboratory.
2.2 Cell culture
Striatal precursor 2a1 cells were cultured under standard conditions in DMEMHG supplemented with 10% (v/v) FCS, 2 mM L-glutamine, penicillin (100 U/mL) and
