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Evaluating the Beneficial and Detrimental Effects of Bile Pigments in Early and Later Life

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The heme degradation pathway has been conserved throughout phylogeny and allows for the removal of a pro-oxidant and the generation of unique molecules including bile pigments with important cellular functions. The impact of bile pigments on health and disease are reviewed, as is the special circumstance of neonatal hyperbilirubinemia. In addition, the importance of promoter polymorphisms in the UDP-glucuronosyl transferase gene (UGTA1), which is key to the elimination of excess bilirubin and to the prevention of its toxicity, are discussed. Overall, the duality of bile pigments as either cytoprotective or toxic molecules is highlighted.
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REVIEW ARTICLE
published: 22 June 2012
doi: 10.3389/fphar.2012.00115
Evaluating the beneficial and detrimental effects of bile
pigments in early and later life
Phyllis A. Dennery
1,2
*
1
Division of Neonatology, Childrens Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
2
Division of Neonatology, Department of Pediatrics, University of Pennsylvania, Philadelphia, PA, USA
Edited by:
Jaime Kapitulnik,The Hebrew
University of Jerusalem, Israel
Reviewed by:
David K. Stevenson, Stanford
University School of Medicine, USA
Michael Kaplan, Shaare Zedek
Medical Center, Israel
*Correspondence:
Phyllis A. Dennery , Division of
Neonatology, Department of
Pediatrics, University of Pennsylvania,
34th and Civic Center Boulevard,
Philadelphia, PA 19104-4318, USA.
e-mail: dennery@email.chop.edu
The heme degradation pathway has been conserved throughout phylogeny and allows for
the removal of a pro-oxidant and the generation of unique molecules including bile pig-
ments with important cellular functions.The impact of bile pigments on health and disease
are reviewed, as is the special circumstance of neonatal hyperbilirubinemia. In addition,
the importance of promoter polymorphisms in the UDP-glucuronosyl transferase gene
(UGTA1), which is key to the elimination of excess bilirubin and to the prevention of its tox-
icity, are discussed. Overall, the duality of bile pigments as either cytoprotective or toxic
molecules is highlighted.
Keywords: neonatal jaundice, kernicterus, UDP-glucuronyltransferase, antioxidant, polymorphisms
INTRODUCTION
The generation of bile pigments occurs through a unique pathway
for the degradation of heme, limited by the enzyme hemeoxy-
genase (HO). This enzymatic reaction requires molecular oxy-
gen (O
2
) and NADPH as a reducing equivalent and results in
the formation of biliverdin and the release of carbon monox-
ide (CO) in an equimolar ratio. In addition, reduction of heme
iron from Fe
2+
–Fe
3+
occurs with the transfer of electrons from
oxygen with NADPH as a source of reducing equivalents. Also,
oxidative cleavage of the α-methene carbon bridge in the heme
molecule forms biliverdin, releases carbon monoxide and iron
(Figure 1). In most circumstances, heme does not accumulate
freely but rather, it is bound to hemo proteins that are essential
for cellular metabolism. An intricate enzymatic cascade regulates
the production of heme (Ryter and Tyrrell, 2000). In patholog-
ical conditions, free heme is released from hemoglobin and can
deposit in tissues because it is lipophilic and can lead to the for-
mation of oxygen radicals therefore, the enzymatic reaction of
HO is essential to preventing this since HO-1 is highly inducible
by the substrate heme or by oxidative stress (Chang et al., 2005).
The constitutive isoenzyme HO-2 can also catalyze the degrada-
tion of heme and is found in high abundance in the brain and
testes (Maines et al., 1986; Maines, 1988). In the last steps of
the reaction, biliverdin reductase (BVR), a microsomal enzyme,
converts biliverdin into bilirubin in a non-rate-limiting fashion
(Figure 1). Bilirubin, unlike biliverdin is not water soluble, but
rather lipophilic, and can penetrate cellular membranes. To be
made water soluble and therefore excretable in the gastrointestinal
tract, it must be conjugated. The latter is regulated by UDP-
glucuronosyl transferase 1A1 (UGT1A1), an enzyme that adds
two glucuronide residues to bilirubin to render it water soluble
(Figure 1).
The byproducts of HO-mediated heme degradation can have
both positive and negative effects on cellular function. These will
be outlined in this review.
BILIRUBIN
EFFECTS OF BILIRUBIN IN THE NEONATE
Newborns have elevated numbers of red blood cells with a short-
ened life-span. When these cells lyse, heme is released from
hemoglobin. In addition, due to a reduced ability to conjugate
bilirubin formed during the degradation of heme, this pigment
can accumulate in the serum in the first days of life leading to
a transient hyperbilirubinemia, which typically resolves within
the first weeks of life. In fact, the average full-term newborn
infant has a peak serum bilirubin concentration of 5–6 mg/dL
(86–103 µmol/L). This level is referred to as physiologic jaundice.
However, in some circumstances, such as increased accumulation
of heme (i.e., birth trauma, bruising, hemolysis), serum bilirubin
levels can increase beyond the physiologic range. If serum biliru-
bin values are between 7 and 17 mg/dL (104–291 µmol/L), this is
then referred to as exaggerated physiologic jaundice. However, this
must be judged according to the infant’s age in hours on the biliru-
bin nomogram, as serum bilirubin levels change rapidly during
the first week of life. Serum bilirubin concentrations higher than
17 mg/dL in full-term infants are considered pathologic and can
be associated with adverse sequelae. Fortunately, most infants will
not be affected until their bilirubin levels are significantly higher
than 17 mg/dL because the toxicity of bilirubin is dictated by many
factors including age (the younger, the more vulnerable), matu-
rity (prematures are more vulnerable), and associated illnesses
(hemolysis, sepsis, acidosis worsen bilirubin toxicity) amongst
other factors. The most severe manifestation of bilirubin toxi-
city is kernicterus, a rare but devastating condition with acute
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Dennery Bilirubin good and bad
FIGURE 1 | Heme degradation pathway. Heme from hemoglobin and
cellular hemo proteins is metabolized in a rate-limiting step by HO. The
reaction utilizes molecular oxygen and NADPH as a reducing equivalent.
This leads to the release of iron, water, and CO as well as biliverdin. The
latter is converted in a non-rate-limiting fashion to bilirubin by BVR. Since
bilirubin is not water soluble, it is conjugated to monoglucuronide and
diglucuronide forms, which can then be excreted. These glucuronide
residues can be removed by intestinal bacteria and allow unconjugated
bilirubin to re-enter the circulation.
neurological abnormalities including seizures, opisthotonus, and
hypertonia and long lasting sequelae including sensorineural deaf-
ness, athetoid cerebral palsy, and delayed motor skills (Dennery
et al., 2001).
Prior to the late 1980s, physicians had a very aggressive
approach to the management of hyperbilirubinemia in neonates,
which included institution of phototherapy at low levels of biliru-
bin and exchange transfusion if the level exceeded 20 mg/dL in all
cases. This approach changed radically since kernicterus was such
a rare condition and, with the advent of Rhogam prophylaxis for
Rh-negative pregnant mothers, kernicterus was thought by some
to be nearly eradicated in developed nations (Watchko and Oski,
1983). In addition, reports of the beneficial, antioxidant effects
of bilirubin (Stocker et al., 1987, 1990; McDonagh, 1990) made
this aggressive approach seem even more unwarranted. Alas, with
this change in attitude, pediatricians saw a resurgence of biliru-
bin neurotoxicity, and were faced with litigation for the negligent
practice of not having taken simple measures to prevent the devas-
tating consequences of hyperbilirubinemia (Maisels and Newman,
2007; Bhutani et al., 1999). In the present day, we have adopted a
more thoughtful approach to this preventable problem by using
strict guidelines for universal screening and by instituting therapy
guided by age-based serum bilirubin thresholds (Bhutani et al.,
1999; Maisels and Newman, 2007).
It is not clear why bilirubin accumulates in the first days of
life and what is its value. Physiologic hyperbilirubinemia is a
phenomenon only seen in mammals. Many have speculated a tele-
ological benefit to this reaction (McDonagh, 1990) and laboratory
investigations do confirm that bilirubin has significant antioxidant
properties both in vitro (Stocker et al., 1987; Mireles et al., 1999;
Granato et al., 2003) and in vivo (Dennery et al., 1995; Mayer,
2000). Perhaps this may be useful in the transition from the rela-
tively hypoxic environment of the wound to ambient air. This is
not yet clear. Also, the exact dose at which bilirubin is toxic to cells
vs. beneficial is also not yet known.
In infants with Rh hemolytic disease, peak serum bilirubin con-
centrations above 20 mg/dL predict poor outcome however, many
infants without an obvious hemolytic etiology for their jaun-
dice are normal at serum bilirubin concentrations of 25 mg/dL
or higher. Looking at this more closely, 8% of infants with Rh-
associated hemolysis and serum bilirubin concentrations of 19–
24 mg/dL had kernicterus whereas this condition was observed
in 73% of infants with concentrations of 30–40 mg/dL demon-
strating the dose effect of bilirubin, at least in hemolytic children.
Bilirubin enters the brain and targets the basal ganglia (Johnston
and Hoon, 2000) and the auditory nerve preferentially (Shapiro
and Nakamura, 2001). This occurs if bilirubin is not bound to
albumin or is unconjugated or if there has been damage to the
blood–brain barrier. As an example,a newborn infant with a serum
albumin concentration of 3 g/dL can bind 25 mg/dL of bilirubin.
In current clinical practice, the bilirubin albumin ratio is taken into
consideration to determine levels at which physicians should pro-
ceed to more aggressive management of hyperbilirubinemia (i.e.,
exchange transfusion). If the serum albumin concentration is low,
the risk of kernicterus increases because free bilirubin enters tissues
and causes its toxic effects (Ahlfors et al., 2009). Conditions that
alter the blood–brain barrier, such as infection, acidosis, hyper-
oxia, sepsis, prematurity, and hyperosmolarity, may also affect the
entry of bilirubin into the brain (Dennery et al., 2001).
MECHANISMS OF TOXICITY OF BILIRUBIN
Bilirubin has high affinity for membrane phospholipids thereby
entering cells and inhibiting mitochondrial enzymes (Chuniaud
et al., 1996; Rodrigues et al., 2002a), interfering with DNA syn-
thesis, inducing DNA-strand breaks (Rosenstein et al., 1983), and
inhibiting protein synthesis and phosphorylation. In the brain,
bilirubin inhibits the uptake of tyrosine, reduces synaptic trans-
mission, and inhibits N -methyl-d-aspartate-receptor ion chan-
nels. Overall, bilirubin can interfere with neuro excitatory signals
and impair nerve conduction particularly in the auditory nerve
(Bratlid, 1990). Unconjugated bilirubin directly interacts with
mitochondria influencing membrane lipid and protein proper-
ties, redox status, and cytochrome c content (Rodrigues et al.,
2002b). It can work in concert with amyloid β peptide to activate
apoptosis in neural cells (Rodrigues et al., 2000). Interestingly,
younger animals are less susceptible to bilirubin-related mito-
chondrial injury (Rodrigues et al., 2002c) and the toxicity of
bilirubin is not restricted to neonates. In patients with Crigler–
Najjar I syndrome and absent activity of the UGTA1, therefore
a complete lack of ability to conjugate bilirubin, the risk of ker-
nicterus is quite high especially in the face of intercurrent illnesses
and these patients require life-long phototherapy and/or liver
transplantation (Strauss et al., 2006).
In the nervous system, the susceptibility of bilirubin varies with
cell type (Ngai et al., 2000). In brain endothelial cells, bilirubin
resulted in apoptosis in a time-dependent manner (Akin et al.,
Frontiers in Pharmacology | Drug Metabolism and Transport June 2012 | Volume 3 | Article 115 | 2
Dennery Bilirubin good and bad
2002). Unconjugated bilirubin induced protein oxidation and
lipid peroxidation and reduced antioxidant defenses in neuronal
cells in culture (Brito et al., 2008). Although toxic to cultured
neuroblastoma cells, exposure to unconjugated bilirubin induced
genes involved in the endoplasmic reticulum stress response in
surviving cells thereby enhancing cellular homeostasis (Calligaris
et al., 2009). In astrocytes, unconjugated bilirubin up-regulated
the multidrug resistance-associated protein-1 and increased its
trafficking to the plasma membrane, thus reducing its cytotoxi-
city by preventing its intracellular accumulation (Gennuso et al.,
2004).
The toxic effects of bilirubin are not limited to the brain.
Unconjugated bilirubin can mediate apoptosis in cultured hepa-
tocytes by increasing oxidative stress and enhancing caspase-9
activity (Oakes and Bend, 2005). In erythrocytes, high biliru-
bin concentrations can induce hemolysis and lead to membrane
disruption, which could theoretically worsen hemolytic anemia
(Brites et al., 1997). We observed that in erythrocytes derived
from cord blood, concentrations of bilirubin equal to or exceed-
ing 30 mg/dL were associated with increased protein oxidation,
decreased erythrocyte glucose-6 phosphate dehydrogenase and
adenosine triphosphatase activity as well as altered cell mem-
brane integrity (Mireles et al., 1999). There was a correlation
between the release of unconjugated bilirubin and hepatotoxic-
ity after TNF-a administration, in mice and this was resolved with
HO inhibitors (Van Molle and Libert, 2003). Another important
cytotoxicity of bilirubin involves its effects on complement acti-
vation, a key element of immune defense. Unconjugated bilirubin
interferes with the interaction between C1q and immunoglob-
ulins, which results in decreased complement activation via the
classical pathway (Basiglio et al., 2010).
ANTIOXIDANT BENEFITS OF BILIRUBIN
Despite the fact that bilirubin may be toxic at higher concentra-
tions, there is still significant evidence that it is a potent antioxidant
at micromolar concentrations in vitro and in vivo. In fact, biliru-
bin is the most abundant cellular antioxidant. In vitro, bilirubin is
a chain-breaking molecule that can scavenge the hydroxyl radical
better than a-tocopherol, a well-known antioxidant against lipid
peroxidation (Stocker et al., 1987; Mireles et al., 1999). Although
incubation with bilirubin and albumin at concentrations greater
than 30 mg/dL was associated with dose-dependent injury in ery-
throcytes derived from cord blood, protection against lipid per-
oxidation was seen at lower concentrations (Mireles et al., 1999),
indicating the duality of bilirubin as a cytotoxic and cytoprotec-
tive molecule. Similarly, hemeoxygenase is both detrimental and
beneficial based on levels of activity. Nevertheless, in that study, no
change in bilirubin levels could be detected to explain the toxicity
of hemeoxygenase (Suttner and Dennery, 1999).
Similarly, in vivo, there are several examples of the beneficial
effects of hyperbilirubinemia. In 50 patients older than 40 years
with Gilbert syndrome, a relatively benign condition leading to
mild to moderate unconjugated hyperbilirubinemia because of
impaired glucuronidation, occurrence of ischemic heart disease
was compared to that of a large cohort of patients without the dis-
ease. Ischemic heart disease occurred in only 2% of the Gilbert
patients compared to 12.1% of the controls and, interestingly,
hyperbilirubinemia rather than elevation of HDL cholesterol lev-
els seemed to be more important in protection from ischemic
heart disease (Vitek et al., 2002). In a case report, resolution of
corticosteroid- and cyclophosphamide-resistant pulmonary fibro-
sis occurred with onset of hyperbilirubinemia due to biliary
obstruction in a patient who developed elevated conjugated biliru-
bin levels (Ohrui et al., 2001), suggesting that higher serum biliru-
bin levels could reverse pulmonary fibrosis. The mechanisms by
which this could occur are not yet explored. In organ transplan-
tation, bilirubin can be protective against graft rejection (Ollinger
et al., 2007). Injection of bilirubin in mouse organ recipients pro-
longed islet allograft survival and induced tolerance induction
and graft acceptance via a regulatory T cell-dependent mech-
anism involving CD4(+) and CD25(+) cells. In fact, bilirubin
enhanced de novo generation of regulatory T cells in the recipients
thereby preventing rejection (Rocuts et al., 2010). Another novel
mechanisms by which bilirubin may be protective is by the regu-
lation of rapid eye movement sleep and by mediating some of the
antidepressant effects of ambient light (Oren, 1997). Whether the
antioxidant effects mediate these benefits is not yet clear.
Overall, the beneficial effects of bilirubin have been demon-
strated in various models but beyond a certain threshold, bilirubin
is clearly toxic.
EPIDEMIOLOGIC EVIDENCE OF BILIRUBIN AS A CYTOPROTECTIVE
MOLECULE
To further understand whether bilirubin is cytoprotective in
humans, epidemiologic studies can provide a clue. Regulation
of bilirubin conjugation is key in the accumulation of bilirubin
and its potential benefits or toxicity, therefore, studies compar-
ing patients with differences in the ability to conjugate biliru-
bin may provide clues. The promoter of the UGT1A1 gene has
regions of TA repeats, which regulate its transcriptional effi-
ciency. In Caucasian populations, an additional TA repeat (TA
7
vs. TA
6
) is necessary but not sufficient to cause Gilbert syndrome
(Bartlett and Gourley, 2011). Strong associations between poly-
morphisms in the UGT1A1 gene and human disease have been
shown. In particular, there have been associations with altered
bilirubin conjugation and the occurrence of various cancers. The
common UGT1A1
28 allele results in elevated plasma bilirubin
levels and is strongly associated with Gilbert syndrome in Cau-
casians. Low serum bilirubin levels observed in a Caucasian cohort
with predicted high activity of UGT1A1 were associated with an
increased risk of esophageal cancer. Interestingly, the UGT1A8
and UGT2B4 genotypes, associated with decreased UGT enzyme
activity and increased unconjugated bilirubin levels, were also
significantly associated with increased risk of esophageal cancer
(Dura et al., 2012). In another study, the UGT1A gene cluster
on chromosome 2q37.1 was identified in a cohort of patients
with bladder cancer suggesting that enhanced UGT1A may pro-
tect from bladder cancer by increasing the removal of carcinogens
from bladder epithelium (Tang et al., 2012). In a meta-analysis of
21 case-control studies cancer risk was associated with intermedi-
ate, and low activity of UGT1A7 genotypes, found predominantly
in Asians (Lu et al., 2011). In contrast to the other studies, the
TA repeat polymorphism of UGT1A1 gene did not alter prostate
cancer risk susceptibility in Caucasian men (Karatzas et al., 2010).
www.frontiersin.org June 2012 | Volume 3 | Article 115 | 3
Dennery Bilirubin good and bad
Overall, these studies suggest that the concentration of bilirubin
in the serum determines whether it is beneficial or detrimental.
Not only does the UGT1 gene play a role in cancer, it appears to
have important effects in other diseases. For example, the homozy-
gous state associated with higher serum bilirubin levels appeared
to be protective against Crohns disease (de Vries et al., 2012).
Serum bilirubin, independent of variation in UGT promoter
activity, is also associated with diseases in large populations, in
particular in cardiovascular disease. In a Swedish cohort, plasma
bilirubin was lower in 231 cases of ischemic stroke than in 462
matched controls but the difference reached significance only in
women (Ekblom et al., 2010). In males with coronary artery dis-
ease, there was inverse association between serum total bilirubin
and coronary artery calcification score. Additionally, bilirubin was
associated with reduced c-reactive protein levels, which could
explain the lower calcification scores (Zhang et al., 2012). In
another study, bilirubin levels were also inversely associated with
the presence of coronary heart disease. Interestingly, bilirubin lev-
els were significantly raised after treatment with 80 mg simvastatin
independent of changes in liver enzymes (Nolting et al., 2011).
Despite the beneficial effects of bilirubin, the biggest challenge
remains determining a specific threshold at which bilirubin is
toxic vs. beneficial. It seems paradoxical that early events in biliru-
bin toxicity may involve increased oxidative stress and changes in
redox status (Tell and Gustincich, 2009) yet conversely, bilirubin
alleviates oxidative stress.
ANTIOXIDANT PROPERTIES OF BILIVERDIN
Although biliverdin does not accumulate in mammals, since
it is rapidly converted to bilirubin through the action of
biliverdin reductase (BVR), it may have important signaling
effects. In macrophages, biliverdin activates endothelial nitric
oxide, resulting in NO-dependent S-nitrosylation of BVR. The
mechanisms by which biliverdin mediates this effect was via
the repression of Toll-like receptor-4 (Wegiel et al., 2011). In
another study, rats injected intraperitoneally with biliverdin before
undergoing lung transplantation had less evidence of inflamma-
tion, oxidative injury, and apoptosis suggesting that biliverdin has
anti-inflammatory and anti-apoptotic effects (Wang et al., 2010).
Despite these data, the most plausible effect of biliverdin is to serve
as a signaling molecule that may regulate BVR (Lerner-Marmarosh
et al., 2008). The properties of this enzyme have been reviewed at
length in a previous issue of this journal (Gibbs et al., 2012).
OTHER POSSIBLE MEDIATORS OF HO-RELATED CYTOPROTECTIVE
EFFECTS
Although the byproducts of the HO-1 reaction are important
cytoprotective molecules, a likely factor that influences the ben-
eficial effects of the HO reaction is the degradation of a potent
oxidant, heme. In one example, induction of HO-1 prevented pho-
todynamic therapy-induced tumor necrosis, but neither bilirubin,
biliverdin nor CO was responsible for this cytoprotection. In fact,
the iron chelator desferrioxamine enhanced the cytotoxic effects
of photodynamic therapy suggesting that heme was key important
to enhancing the tumor killing effects of this therapy (Nowis et al.,
2006).
SUMMARY
Overall, the byproducts of the HO reaction are important cytopro-
tective molecules that have clinically significant effects in various
diseases. Nevertheless, in most cases, these molecules can also be
cytotoxic under specific circumstances and/or at high concentra-
tions. Potential therapeutic interventions will need to balance the
potential benefits with the risk of toxicity to be most effective.
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Conflict of Interest Statement: The
author declares that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 10 April 2012; paper pend-
ing published: 23 April 2012; accepted:
29 May 2012; published online: 22 June
2012.
Citation: Dennery PA (2012) Evalu-
ating the beneficial and detrimental
effects of bile pigments in early and
later life. Front. Pharmacol. 3:115. doi:
10.3389/fphar.2012.00115
This article was submitted to Frontiers
in Drug Metabolism and Transport, a
specialty of Frontiers in Pharmacology.
Copyright © 2012 Dennery. This is an
open-access article distributed under the
terms of the Creative Commons Attribu-
tion Non Commercial License, which per-
mits non-commercial use, distribution,
and reproduction in other forums, pro-
vided the original authors and source are
credited.
www.frontiersin.org June 2012 | Volume 3 | Article 115 | 5
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The aim of our meta-analysis was to assess the association between UGT1A7 polymorphisms and cancer risk. Case?control studies containing available polymorphic alleles (UGT1A7*1,*2,*3, and*4) and genotypes categorized according to enzymatic activity (High, Intermediate, and Low) were chosen to assess this association. Twenty-one case?control studies were identified. Meta-analysis indicated that UGT1A7 had a significant effect on cancer risk. In subgroup analysis, a significantly increased risk was associated with East Asians, hepatocellular cancer, and colorectal cancer. This meta-analysis suggested that there is a cancer risk associated with UGT1A7*3, Intermediate, and Low activity UGT1A7 genotypes, which is most evident in Asian individuals.