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Current Pharmaceutical Design, 2009, 15, 2908-2914

Redox State, Oxidative Stress, and Molecular Mechanisms of Protective


and Toxic Effects of Bilirubin on Cells
Gianluca Tell1,* and Stefano Gustincich2
1

Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy and 2Sector of Neurobiology, International School for Advanced Studies (SISSA), AREA Science Park, s.s. 14, Km 163.5, Basovizza, 34012
Trieste, Italy
Abstract: Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. UCB is a potent antioxidant molecule as well as an indirect pro-oxidant generator. Growing evidence suggests that its major cellular effects
are mediated by inhibiting proliferation in cancer cell lines and eliciting cytotoxicity, particularly in neurons and glial
cells. Here we describe studies showing that alteration of the redox status and generation of oxidative stress are likely
early events responsible for UCB-induced cytotoxicity. We then elucidate some of the molecular pathways that govern
these effects.

Key Words: Bilirubin/oxidative stress/cytotoxicity.


BIOLOGICAL EFFECTS OF BILIRUBIN
Unconjugated bilirubin (UCB), the major degradation
product of heme, has long been considered to be a useless
waste product. Studies during the last two decades, however,
have revealed its dual natural functions, acting as a boon at
physiological and mildly elevated concentrations and as a
curse at higher, clinically relevant concentrations, especially in the central nervous system [1-3]. Some neonates
with severe hyperbilirubinemia develop bilirubin encephalopathy, in which the multifocal deposition of UCB in selected
regions of the brain results in temporary or permanent impairment of auditory, motor or mental functions [4]. Recent
increases in the prevalence of bilirubin encephalopathy and
its occasional occurrence at plasma bilirubin levels below
therapeutic guidelines have revived interest in understanding
the mechanisms of UCB-induced neurotoxicity [5].
In the current model three factors are believed to play a
crucial role in Bilirubin Induced Neurological Dysfunction
(BIND): the plasma concentration of unbound UCB (Bf), the
intracellular concentration of UCB and the molecular
mechanisms that limit intracellular accumulation of UCB.
Since unbound UCB freely diffuses across cell membranes, a first requirement for neurotoxicity is a high Bf in
plasma [5-7]. However, it is clear that a high Bf cannot, per
se, predict the occurrence or severity of brain damage. A
review of studies of in vitro exposure of neurons and astrocytes to UCB has revealed cytotoxicity at Bf concentrations
in the medium above aqueous saturation (70 nM) [8] indicating a link between direct exposure to high Bf and neuronal
dysfunction [9]. Interestingly, neurotoxicity is exacerbated,
and occurs at lower Bf, when ATP-dependent cellular export

*Address correspondence to this author at the Department of Biomedical


Sciences and Technologies, University of Udine, 33100 Udine, Italy; Tel:
+39 0432 494311; Fax: +39 0432 494301; E-mail: gianluca.tell@uniud.it

1381-6128/09 $55.00+.00

of UCB by Multidrug resistance-associated protein 1


(MRP1/Mrp1) is inhibited [10] or genetically deleted [11].
These in vitro experiments prove that: a) supersaturation of
the medium with Bf is not essential for neurotoxicity; and b)
the intracellular UCB concentration is the determinant of
toxicity, and is regulated by the balance between diffusion
gradients (determined by extracellular Bf levels) and export
mechanisms at the cell membrane.
The molecular mechanisms underlying or contributing to
UCB cytotoxicity are not completely understood. Beginning
in 1999, it was shown that UCB is a pro-apoptotic agent,
suppressing cell growth by inducing DNA fragmentation,
mitochondrial release of cytochrome c, activation of caspase3 and cleavage of poly(ADP)ribose polymerase [9, 12-17].
More recently, oxidative stress has emerged as a potential
crucial event [18]. In various cellular systems, UCB causes
ROS production, protein oxidation, lipid peroxidation and
disruption of glutathione metabolism [18, 19]. Oxidative
stress generation mirrors UCB-mediated apoptosis [15] and
UCB-mediated oxidative stress is also in part responsible for
its inhibition of cell growth [20]. We here summarize evidence for a crucial role of oxidative stress, and its initiation
by UCB, in the mechanisms causing BIND.
OXIDATIVE STRESS HOMEOSTASIS
Oxidative stress results from an imbalance between the
generation of reactive oxygen species (such as H2O2, OH,
O2-, collectively known as ROS, Fig. 1) and the antioxidant
defence capacity of the cell [21], affecting major cellular
components, including lipids, proteins, and DNA. This phenomenon is closely associated to a number of human disorders including cardiovascular diseases, diabetes, cancer and
neurodegenerative disorders [22-24]. ROS cause extensive
cellular damage but can also play important physiological
functions. Cells are, therefore, provided with efficient molecular strategies to strictly control the intracellular ROS

2009 Bentham Science Publishers Ltd.

Bilirubin and the Cellular Redox State

level and to maintain the balance between oxidant and antioxidant molecules.

Current Pharmaceutical Design, 2009, Vol. 15, No. 25

2909

ily. This oxidative-dependent cellular signalling pathway


may then lead to apoptosis.
Among the major players involved in the homeostatic
response, the anti-oxidant Parkinsons disease associated DJ1 protein acts as an atypical peroxiredoxin-like peroxidase
and as a redox-regulated chaperonin while the carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin ligase/cochaperone [27, 28].

Fig. (1). Major types of Reactive Oxygen Species (ROS) and their
derivatives.

In non-phagocytic cells mitochondria are the major


source of cellular ROS during the respiratory process (Fig.
2). However, ROS may be also produced by membraneassociated enzymes such as NADPH-oxidases. Low levels of
potentially toxic oxygen metabolites are physiologically
generated in cells and tissues during oxidative phosphorylation [25]. The resulting moderate levels of ROS play an essential role in the modulation of several physiological functions of the cell, including gene expression and signal transduction. Under normal conditions, it is estimated that up to
1% of the mitochondrial electron flow primarily leads to the
formation of superoxide anion that is generated by the univalent reduction of molecular oxygen. This process is mediated
by enzymes such as NADP(H) oxidases and xanthine oxidase (Fig. 2, reactions a and b) or non-enzymatically by redox-reactive compounds, such as the semi-ubiquinone compound of the mitochondrial electron transport chain (Fig. 2,
reaction c). Interference with electron transport can dramatically increase superoxide production. Superoxide dismutases
(SODs) catalyze dismutation of two superoxide anions into
hydrogen peroxide and molecular oxygen [26] (Fig. 2, reaction d). H2O2 can undergo the Fenton reaction with reduced
transition metals, to produce the highly reactive hydroxyl
radical (OH) (Fig. 2, reaction e), a far more damaging
molecule. Alternatively, H2O2 may be converted into water,
and thus inactivated, by the enzymes catalase (CAT) and
glutathione peroxidase (GPX) (Fig. 2, reactions f and g).
During the GPX reaction, glutathione is oxidized to glutathione disulfide, which can then be reconverted to glutathione by glutathione reductase in an NADPH-consuming
process (Fig. 2, reaction h).
Cellular oxidative stress response involves the modulation of the activity of large sets of proteins. These are modified by ROS on aminoacids targets like tryptophan, tyrosine,
histidine and, most importantly, cysteine. ROS-mediated
protein modifications can then lead to the binding of chaperonins that induce protein degradation through the UPS
and/or autophagy. They may also stimulate cellular kinases
such as the mitogen-activated protein kinase (MAPK) fam-

Importantly, ROS may also influence gene expression


through different molecular mechanisms leading to transcription factors activation. Nuclear factor-erythroid-2-related
factor 2 (Nrf2) plays a crucial role in the coordinated induction of antioxidant response element (ARE)-bearing genes,
including those encoding endogenous antioxidants, phase II
detoxifying enzymes, and transporters. In resting cells, Nrf2
is sequestered in the cytoplasm as an inactive complex with
the repressor Kelch-like ECH-associated protein 1 (Keap1).
The release of Nrf2 from its repressor is achieved by oxidation or covalent modification of some critical cysteine thiols
in Keap1 sequence [29].

Fig. (2). Generation of ROS in mitochondria, the major source


of cellular ROS in non-phagocytic cells. Under physiological
conditions, low levels of potentially toxic ROS, generated in cells
during oxidative phosphorylation play an integral role in the modulation of cellular gene expression, signal transduction, and defence
against invading pathogens. Under normal conditions, it is estimated that up to 1% of the mitochondrial electron flow leads to the
formation of superoxide anion by the univalent reduction of molecular oxygen (O2). This process is mediated by enzymes such as
NADP(H) oxidases (reaction a) and xanthine oxidase (reaction b)
or nonenzymatically by redox-reactive compounds, such as the
semi-ubiquinone compound of the mitochondrial electron transport
chain (reaction c). Interference with electron transport can dramatically increase superoxide production. Superoxide is rapidly converted to H2O2 and O2 by the superoxide dismutases (SODs) (reaction d). H2O2 can react with reduced transition metals (Fe2+ & Cu+),
via the Fenton reaction (e), to produce the more highly reactive
hydroxyl radical, OH. Alternatively, H2O2 may be converted into
water by the enzymes catalase (CAT, reaction f ) and glutathione
peroxidase (GPX, reaction g). During the glutathione peroxidase
reaction, glutathione (GSH) is oxidized to glutathione disulfide
(GSSG), which can be the reconverted to glutathione by GSH reductase in an NADPH-consuming process (reaction h).

The redox status of reactive Cys residues may also control the transcriptional activity of the TFs itself for its loca-

2910 Current Pharmaceutical Design, 2009, Vol. 15, No. 25

tion within the DNA-binding domain. Furthermore, APE1


has been identified as a protein able to induce the DNA binding activity of several transcription factors in a redoxdependent manner [30, 31].
BILIRUBIN AS AN ANTIOXIDANT: CHEMICAL
AND IN VITRO BIOCHEMICAL STUDIES
In 1954 Bernard and colleagues showed for the first time
that UCB has antioxidant properties [32]. They proved that
small amounts of UCB prevented the autooxidation of
vitamin A as well as the ultraviolet light-induced autooxidation of linoleic acid and other unsaturated fatty acids [33].
These pioneering studies were later confirmed by evidences
that bilirubin is an efficient scavenger of singlet oxygen [3437]. It also reacts with superoxide anion [38] and serves as a
substrate for peroxidases in the presence of hydrogen
peroxide or organic hydroperoxides [39].
Bliuger et al. [40] and Stocker and coworkers then proved
that bilirubin, its polar conjugates, and its dehhydrogenated
derivative, biliverdin, were able to physiologically limit
oxidation of fatty acids or their derivatives [2, 41-44], even
when bound to plasma albumin. Wu et al. [45] showed that
UCB is 20 times more effective in preventing LDL oxidation
than the vitamin E analogue Trolox. Additionally, bilirubin
and/or its conjugates were found effective in protecting
albumin from oxidative damage, in strongly inhibiting the
formation of protein carbonyls [46], in reducing chemicallygenerated peroxyl radicals in homogenous solutions and in
multilamellar liposomes [2] as well as in protecting human
LDL from oxidation [47-49]. The antioxidant properties of
bile pigments were further confirmed by the studies of
Farrera et al. [50].
The physiological relevance of the antioxidant property
of bilirubin and biliverdin was subsequently demonstrated.
These pigments prevent oxyradical damage to human
ventricular myocytes [51], vascular smooth muscle cells
[52], rat hepatocytes and human erythrocytes [53], and limit
cell damage in rat hearts exposed to ischemia [54]. They also
ameliorated the effects of bleomycine-induced pulmonary
fibrosis [55], as well as intestinal ischemia-reperfusion injury
in rats [56].
Importantly, bilirubin concentrations as low as 10 nmol/l
were found to be effective in protecting neuronal cultures
from the oxidative stress induced by 10,000 times higher
concentrations of hydrogen peroxide. This was attributed to
the biliverdin-bilirubin redox cycle, in which UCB is continuously regenerated from biliverdin by cytosolic biliverdin
reductase. This enables such low concentrations of UCB to
protect against vastly higher concentrations of hydrogen
peroxide [57]. Bilirubin also protects neurons against oxidative stress injury when generated by activation of heme
oxygenase [58].
BILIRUBIN AS AN ANTIOXIDANT: IN VIVO ANIMAL
AND HUMAN STUDIES
Gunn rats, which congenitally lack bilirubin UDP-glucuronosyltransferase and are thus unable to conjugate bilirubin
with glucuronic acid [59, 60], confirmed an effect of elevated UCB levels on redox state. Compared to non-jaundiced
littermates, hyperbilirubinemic Gunn rat pups showed less

Tell and Gustincich

oxidative injury as well as lower concentrations of lipid


peroxides, conjugated dienes and carbonyl proteins [59].
Attenuation of the pressor and prooxidant effects of angiotensin II was also reported in the jaundiced pups, presumably
due to improved scavenging of superoxide anion by high
UCB levels [60]. The physiologic importance of these
antioxidant properties of UCB is demonstrated by it protective effect in: balloon injury-induced neointima formation
[61]; ischemia/reperfusion injury to the isolated rat heart [54]
and liver transplantation [62], in which oxidative stress
damages the donor liver.
CONTRIBUTION OF BILIRUBIN TO TOTAL
ANTIOXIDANT CAPACITY OF SERUM/PLASMA
UCB constitutes approximately 10% of the total antioxidant capacity in normobilirubinemic adults [63]. Both in
vitro and in vivo studies have shown that UCB levels are
directly correlated with the total antioxidant capacity of
serum or plasma. This was documented in a study showing
an increase in total serum antioxidant capacity of the higher
bilirubin concentrations in serum from individuals with
Gilbert syndrome, and in normal serum enriched with bilirubin in vitro [64]. Removal of bilirubin by exchange transfusion diminished the serum antioxidant capacity in term [65]
and premature [66] human neonates, as well as in children
with sickle cell anemia [67]. Further studies [68, 69] found a
substantially lower plasma oxidazibility in hyperbilirubinemic neonatal plasma than in normobilirubinemic adults.
However, contradictory data have been reported [70, 71].
BILIRUBIN AS AN ANTIOXIDANT: OXIDATIVE
STRESS MARKERS IN VIVO
Bilirubin oxidation is apparently one of the major mechanisms by which bilirubin protects against the deleterious
effects of oxidative stress. Bilirubin production and oxidation were increased in the CSF of patients with cerebral
vasospasm after subarachnoid hemorrhage [72]. In concord,
a physiological increase in plasma bilirubin concentrations
occured in adults undergoing intensive physical training who
are known to be relatively resistant to protein oxidation [73].
When UCB highly reacts with ROS, oxidized derivatives
of UCB are formed and excreted in the urine [74]. These
tripyrrolic compounds are called biotripyrrines or biopyrrins.
The ratio of urinary biopyrrins to serum bilirubin is now
considered to be a marker of oxidative stress. Biopyrrin
concentrations correlate with: levels of 8-hydroxydeoxyguanosine (8-OdG), a marker of oxidative DNA damage [75,
76]; acrolein-lysine adducts, a marker of lipid peroxidation
[75, 77]; and with nitrites, which are markers of nitric oxide
production [77].
It is therefore not surprising that increased urinary
excretion of biopyrrins has been found in various pathologic
conditions involving oxidative stress. These include: acute
psychological stress [77], surgical stress [78, 79], sepsis [80],
coronary heart disease [81, 82], congestive heart failure [83],
atopic dermatitis [84], meningitis [84], schizophrenia and
depressions [85], Alzheimers disease [86], and chronic
exposure to asbestos [87]. Interestingly, subjects with Gilbert
syndrome, who have low levels of oxidative stress, excrete
low levels of urinary biopyrrins [88].

Bilirubin and the Cellular Redox State

NEW MOLECULAR PERSPECTIVES FOR THE


DUAL FUNCTIONAL NATURE OF UCB. ROLE OF
APE1/REF-1 IN CELLULAR RESPONSE TO UCB
TOXICITY
In the case of UCB-induced cell toxicity, mitochondria
seem the major site of ROS production, as demonstrated by
the reduction of the mitochondrial membrane potential
(m) upon UCB treatment [15, 19], leading to caspasemediated apoptosis [12, 15-17]. Since UCB pre-treatment
stimulates cell resistance to a further oxidative burst [89],
some of the antioxidant function of this molecule may involve a cellular adaptive response mechanism, implying
transcriptional activation processes.
The cellular response to oxidative stress is a highly regulated process in which the Apurinic Apyrimidinic Endonuclease/Redox effector factor-1 (APE1/Ref-1) protein plays a
central role [30, 31]. APE1/Ref-1 is a ubiquitous, multifunctional protein that possesses both DNA repair and transcriptional regulatory activities in response to oxidative stress
conditions. In particular, APE1/Ref-1 is important in modulating the DNA binding of a number of transcription factors
including AP-1, NF-B and the Early growth response protein-1 (Egr-1) [30, 31].
Activation of APE1/Ref-1 by oxidative stress is exerted
by two independent mechanisms: a) early relocalization from
the cytoplasm to the nucleus; and b) a subsequent increase in
protein expression through a process requiring transcriptional activation [30, 31, 90, 91]. Nuclear accumulation, in
turn, triggers activation of several transcription factors, such
as Egr-1 and p53. Egr-1 is involved in the regulation of cell
growth in response to extracellular signals, such as mitogens
and growth factors, as well as during the cellular response to
oxidative stress [92, 93]. Studies of gene expression in human tumor cells and tissues indicate that Egr-1 exhibits
prominent tumor suppressor functions [94]. This is mediated
by upregulation of the expression of target genes that lead to
apoptosis and inhibition of cell proliferation, such as p53
and the Phosphatase and Tensin homolog, (PTEN) [95].
PTEN is a lipid phosphatase acting as an off switch in the
phosphoinositide 3-kinase/Akt mitogenic signaling pathway
involved in the regulation of cell growth and survival in
several cell lines [96]. Thus, induction of PTEN expression
and/or activity leads to inhibition of cell growth and potentially to apoptosis. We previously observed that the PTEN
promoter is regulated by Egr-1 in an APE1/Ref-1-dependent
manner [90], suggesting the existence of a functional relationship between these three partners in regulating cellular
response to oxidative stress leading to growth suppression.
We recently reported that UCB-induced arrest of cellgrowth, through Akt inhibition, is dependent on increased
ROS generation, mediated by sequential activation of
APE1/Ref-1, Egr-1 and PTEN. This pathway is activated at
a Bf of 80 nM, only slightly above the cytotoxic threshold
for various cell types [8, 9], suggesting it is involved in the
cytotoxicity induced by physiologically relevant concentrations of unbound UCB [23]. APE1/Ref-1 activation thus
appears to be part of a complex network of adaptive cellular
events that determines whether cell death or survival results
from exposure to a diverse range of UCB concentrations
(Fig. 3) [30, 31, 97, 98]. The timing, duration and extent of

Current Pharmaceutical Design, 2009, Vol. 15, No. 25

2911

APE1/Ref-1 activation and ROS production will thus mirror


the toxic or protective effects of UCB on cellular physiology.
UCB can induce the adaptive antioxidant responses at the
same low Bf levels that induce oxidative stress. Interestingly,
activation of the UCB  ROS  APE1/Ref-1  Egr-1 
PTEN signaling pathway and cytotoxicity are antagonized
by blocking ROS formation with the antioxidant, Nacetylcysteine [20]. Higher levels of Bf may be toxic because
they further increase the oxidative stress without enhancing
the adaptive cellular antioxidant system.
These findings suggest that APE1/Ref-1  Egr-1 
PTEN pathway is an important contributor to the cytotoxic
effects produced by modestly increased levels of unbound
bilirubin. An improved understanding of these early molecular events may potentially provide new approaches for the
treatment and prevention of BIND.
Since the initial evidence of the dual role of UCB in cellular homeostasis of oxidative stress, new knowledge has
been accumulated for understanding its molecular basis. This
is important for the description of potential therapeutic effects of high bilirubin in human disease as well as for the
identification of new therapeutics treatments that may interfere with the devastating effects of BIND.

Fig. (3). Proposed model for the central role of APE1 in the mechanisms of cell response to UCB-induced oxidative-stress.

ACKNOWLEDGEMENT
We thank all the members of GT and SG laboratories for
discussion. The financial supports from Telethon - Italy
(grant GGP 05062), from MIUR (FIRB Prot. RBRN07BMCT) and from the Regione Friuli Venezia Giulia to GT
are gratefully acknowledged.
ABBREVIATIONS
AP-1

= Activator Protein-1

APE1/Ref-1 = Apurinic Apyrimidinic Endonuclease/Redox


effector factor-1

2912 Current Pharmaceutical Design, 2009, Vol. 15, No. 25

Bf

= Free unconjugated bilirubin

CAT

= Catalase

Egr-1

= Early growth response protein-1

GPX

= Glutathione peroxidase

LDL

= Low Density Lipoproteins

Mrp1

= Multidrug resistance-associated protein 1

NAC

= N-Acetylcysteine

NF-B

= Nuclear factor -B

PTEN

= Protein Phosphatase and Tensin homolog

ROS

= Reactive oxygen species

SODs

= Superoxide dismutases

UCB

= Unconjugated bilirubin

Tell and Gustincich


[17]

[18]

[19]

[20]

[21]
[22]
[23]
[24]

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