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Review Article
Xanthine Oxidoreductase-Derived Reactive Species:
Physiological and Pathological Effects
Maria Giulia Battelli, Letizia Polito, Massimo Bortolotti, and Andrea Bolognesi
Alma Mater Studiorum-University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine (DIMES),
General Pathology Unit, Via S. Giacomo 14, 40126 Bologna, Italy
Correspondence should be addressed to Letizia Polito; letizia.polito@unibo.it
Received September ; Accepted November
Academic Editor: Tanea T. Reed
Copyright © Maria Giulia Battelli et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Xanthine oxidoreductase (XOR) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid
and is widely distributed among species. In addition to this housekeeping function, mammalian XOR is a physiological source of
superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of variouspathways.
is review intends to address the physiological and pathological roles of XOR-derived oxidant molecules. e cytocidal action of
XOR products has been claimed in relation to tissue damage, in particular damage induced by hypoxia and ischemia. Attempts
to exploit this activity to eliminate unwanted cells via the construction of conjugates have also been reported. Moreover, dierent
aspects of XOR activity related to phlogosis, endothelial activation, leukocyte activation, and vascular tone regulation, have been
taken into consideration. Finally, the positive and negative outcomes concerning cancer pathology have been analyzed because
XOR products may induce mutagenesis, cell proliferation, and tumor progression, but they are also associated with apoptosis and
cell dierentiation. In conclusion, XOR activity generates free radicals and other oxidant reactive species that may result in either
harmful or benecial outcomes.
1. Introduction
e enzyme xanthine oxidoreductase (XOR) has a wide
distribution throughout living organisms and is highly con-
served in prokaryotic, plant, and animal species (reviewed in
[]). XOR is a dimeric metalloavoprotein comprising two
identical subunits of approximately kDa each, including
one molybdenum-containing molybdopterin cofactor (Mo-
co) and one avin adenine dinucleotide (FAD) cofactor, as
well as two nonidentical iron-sulfur redox centers. e purine
oxidation occurs at the Mo-co site, while the FAD site is the
oxidized nicotinamide adenine dinucleotide (NAD+)andO
2
reduction sites. e electron ux moves between the Mo-
co and FAD cofactors through the two iron-sulfur clusters
(reviewed in []).
XOR catalyzes the oxidation of hypoxanthine to xanthine
andxanthinetouricacid,whicharethelasttwostepsof
purine catabolism in the highest primates. XOR has the
rate-limiting function of generating irreversible products,
thus precluding the salvage pathway of purine nucleotides.
Additionally, dierent endogenous metabolites and various
xenobiotics can be oxidized by XOR. Uric acid and its
oxidized derivatives may exert prooxidant activity, mainly
within the cell; however, it has in vivo antioxidant activity,
mainly in body uids. is scavenger action is supposed to
provide an evolutionary advantage to primates that lost their
uricaseactivityviamutationandacquiredacrucialdefense
against oncogenesis by free radicals [].
XOR is highly regulated at both the transcriptional and
posttranslational levels. XOR activity is present in all mam-
maliantissueanduids,although,inmostofthem,itis
expressed at very low levels because the human XOR gene
is usually subjected to a repressing regulation at the tran-
scriptional level []. e highest XOR levels are expressed
in liver, intestine, kidney, and lactating mammary gland
epithelial cells and in vascular endothelial cells (reviewed in
[]). XOR expression may be increased by various stimuli,
such as hormones, growth factors, inammatory cytokines,
and low oxygen tension. At the posttranslational level, XOR
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Oxidative Medicine and Cellular Longevity
Volume 2016, Article ID 3527579, 8 pages
http://dx.doi.org/10.1155/2016/3527579
Oxidative Medicine and Cellular Longevity
is modulated with both quantitative and qualitative changes
initsactivity.XORproteinmaybeproducedindemolybdo-
and/or desulfo-forms, which are inactive in xanthine catalysis
at the Mo-co site, although they can oxidize the reduced
nicotinamide adenine dinucleotide (NADH) at FAD site.
ese defective XOR forms are present in varying percentages
in milk and could be reactivated with the reinsertion of the
lackingatomsattheactivesite.XORactivitywasobservedto
increase in response to hypoxia without changes in the levels
of mRNA or enzyme protein, indicating a posttranslational
regulation of XOR (reviewed in []). However, the most
peculiar modulation of XOR activity in mammals consists of
the conversion from the dehydrogenase to the oxidase form.
is transition occurs in various pathological conditions
(reviewed in []).
In all organisms, XOR is present in its constitutively active
dehydrogenase form, whereas, only in mammals, the NAD+-
dependent xanthine dehydrogenase (XDH, EC ...) can
be converted to the oxidase form (XO, EC ...) through
sulydryl group oxidation or limited proteolysis []. XO
delivers electrons directly to molecular oxygen (O2), thus
generating the reactive oxygen species (ROS), superoxide
anion (O2∙−), and hydrogen peroxide (H2O2),viaaone-
electron and a two-electron reduction, respectively. is gives
rise to the hydroxyl radical (HO∙) in the presence of iron
via the Haber-Weiss and Fenton reactions. e percentage
of divalent versus univalent electron transfer to O2and the
relative quantities of O2∙−and H2O2generated by XO are
dependent upon O2tension, pH, and purine concentration.
us, under normal physiological conditions, H2O2is the
major reactive product derived from the XO-catalyzed O2
reduction. H2O2formation is further favored when both
the O2levels and pH are reduced, such as under ischemic
and/or hypoxic conditions (reviewed in []). Under hypoxic
conditions, these ROS can also be produced by XDH, which,
at the FAD site, can oxidize NADH. Hypoxia-mediated acidic
pH and low O2tension lessen the nitric oxide (NO) formation
by NO synthase and increase its potential to uncouple and
produce O2∙−. ese conditions reduce XOR anity for
xanthine while increasing anity for nitrites, which compete
with xanthine at the Mo-co site and can be reduced to NO.
Under the same conditions the amount of O2∙−formation
by XOR is sucient to react with NO and generate reactive
nitrogen species (RNS), particularly peroxynitrite (ONOO−).
Both free radicals, such as O2∙−,HO
∙, and NO, and nonrad-
ical forms, such as H2O2and ONOO−,haveanoxidizing
eect, thereby contributing to oxidative stress (reviewed in
[]).
e generation of these oxidants may be only partially
blocked by allopurinol, which inhibits the Mo-co site in a
competitive manner but does not inhibit the catalytic activity
at the FAD site. All together, these products are responsible
for XOR cytotoxic and proinammatory activities and for
pro- and antitumorigenic eects, in both physiological and
pathological conditions. e various XOR functions are
dependent on (i) the level of ROS production, as in the case
of cytotoxic eects; (ii) the type of the prevalent product,
for instance, NO in the presence of high nitrate level; (iii)
the specicity of dierent cell types, such as phagocytes
in inammation; (iv) the level of XOR gene expression, in
particular in cancer.
2. Cytotoxicity of Xanthine
Oxidoreductase Products
XOR cytotoxicity received much attention during the second
half of last century, together with the circumstances of the
conversion from XDH to XO. An elevated XO/XDH activity
ratio has been reported in dierent pathological conditions,
which were characterized by tissue damage and cell necrosis.
Inparticular,theXDHtoXOshiwasobservedinavariety
of hypoxic/ischemic conditions (reviewed in []), including
organ transplantation (reviewed in []). In such circum-
stances, any reoxygenation/reperfusion could increase the
supply of oxygen for the formation of oxidants, but it was not
strictly required. Additionally, the conversion from XDH to
XO was not necessary for ROS generation, as discussed above,
especially in the presence of low oxygen tension that favors
the NADH oxidase activity of XOR. However, the formation
of XOR-derived ROS was indicated as the causal agent of the
injury or, at least, of the damage amplication, although more
thanonesourceofROScouldbeimplicated(reviewedin
[]).
e mechanism of ROS cytotoxicity is attributed to
peroxidation of membrane lipids, DNA damage, and protein
oxidation, which impair mitochondrial function and lead to
apoptosis (reviewed in []) (Figure (a)). Indeed, DNA dam-
age and the consequent loss of cloning eciency occurred
in a Burkitt lymphoma-derived cell line via XOR activity
through the production of ROS []. Apoptosis and necrosis
were induced to proliferating human lymphocytes by XOR-
derived oxidative stress, which was prevented by catalase [].
Additionally, oxidative DNA damage, consequent to the ROS
generated by XOR activity, provoked cell death in a nasopha-
ryngeal carcinoma cell line []. Accordingly, XOR-derived
ROS caused DNA double-strand breaks that were associated
with p function/expression and caspase-dependent apop-
tosis in primary human lung microvascular endothelial cells
that were exposed to cigarette smoke extract [].
e oxidative stress could be utilized to eliminate un-
wanted cells, particularly cancer cells. An attempt to take
advantage of the cytotoxicity of XOR products was performed
by conjugating the XOR protein to monoclonal antibodies,
with the intent of delivering XOR activity to the antigen-
bearing cell. XOR-containing conjugates recognizing B lym-
phocyte antigens were prepared with the purpose of autol-
ogous bone marrow graing. ese conjugates selectively
killed B lymphoma cell lines [] without reducing normal
myeloid clonogenic eciency [] and were eective in
bone marrow purging from malignant B lymphocytes [].
XOR immunotargeting was also studied in an experimen-
tal model to eliminate T lymphocytes from bone marrow
for heterologous transplantation []. e cytotoxicity and
selectivity of conjugated XOR were enhanced by the addition
of chelated iron that potentiates the free radical formation
(reviewed in []). e ecacy of XOR activity was proven
in conditions that were very similar to the ex vivo treatment
for bone marrow purging from multiple myeloma cells, with
Oxidative Medicine and Cellular Longevity
Apoptosis
ROS
XOR
Membrane
Protein
DNA
(Necrosis)
(a)
Antibody
PEG
Hematological tumors Solid tumors
Specic delivery Enhanced delivery
(b)
F : Pharmaceutical applications of xanthine oxidoreductase (XOR) cytotoxicity. (a) Mechanisms of ROS cytotoxicity: ROS induce
peroxidation of membrane lipids, DNA damage, and protein oxidation and lead to cell death, mainly via apoptosis through impaired
mitochondrial function (reviewed in []). (b) XORwas conjugated to carriers for the experimental elimination of specic target cells. Selective
cell killing was obtained by conjugating XOR to an antibody that was able to specically deliver reactive oxygen species (ROS) to target cells
[]. Enhanced ROS delivery to solid tumors was achieved by XOR conjugation to polyethylene glycol (PEG) [].
a XOR/antibody conjugate or with a free monoclonal anti-
body followed by a XOR/anti-antibody conjugate. Both direct
and indirect methods induced a prevalence of apoptotic death
over necrosis in malignant B lymphocytes [] (Figure (b)).
To improve the ROS delivery eciency to solid tumors,
XOR was conjugated to polyethylene glycol [], which (i)
confers superior in vivo pharmacokinetic characteristics by
increasing the blood half-life of the enzyme; (ii) counteracts
the aspecic adhesiveness of XOR to the vascular inner sur-
face; and (iii) concentrates XOR in cancer tissues by exploit-
ing the enhanced permeability and retention eect of macro-
molecules and lipids in solid tumors (reviewed in [])
(Figure (b)).
3. Proinflammatory Activity of Xanthine
Oxidoreductase Products
e evolution of XOR from the highly conserved dehydroge-
nase to the interconvertible mammalian oxidase form confers
to its enzyme activity a new role of producing physiologic
signal transduction that is mediated by ROS as secondary
messengers (reviewed in [, ]).
XOR activity is known to be upregulated in response
to inammatory cytokines [], which induce the XDH to
XO transition and also increase the XOR level in plasma
[], supporting the hypothesis that XOR is a component of
the innate immune system (reviewed in []). Indeed, XOR
has been implicated in the defense against infectious diseases
because of its capability of activating the cellular phlogistic
response at various levels (reviewed in []). XOR-derived
ROS promote leukocyte-endothelial cell interactions by
increasing the adhesion of phagocytes []. ey also induce
the production of cytokines [], thus amplifying the inam-
matory response, and chemotactic factors [], which cause
the accumulation of polymorphonuclear granulocytes in the
microvasculature []. e bactericidal activity of XOR may
contribute to the oxygen-dependent cell killing during leuko-
cyte phagocytosis through ROS and ONOO−production
[]. e antibacterial properties of XOR suggest that its
abundanceinmilkcouldhavetheroleofanaturalantibiotic,
representing one of the reasons to encourage breastfeeding by
mothers [] (Figure (a)).
e usually very low XOR serum level in humans may
become more elevated in pathological circumstances that
causetissuedamageandthereleaseofXORfromcells
into the bloodstream. Circulating XOR is converted to the
oxidase form and binds to endothelial cells, even at distant
sites, inducing proinammatory signaling or even remote
organ injury (reviewed in []). e proinammatory activity
exerted by the XOR-derived ROS may aect the microvascu-
lar lining by inducing endothelium permeabilization, which
begins both the physiological cascade of immune response
and the pathological events that induce atheromatous
plaque formation (reviewed in []) (Figure (b)). e XOR
Oxidative Medicine and Cellular Longevity
Inammation
Angiogenesis
Tissue repair
ROS
Adhesion
Chemotaxis
XOR
Cytokine release
ROS/RNS production
RNS
Endothelial activation
Leucocyte activation
Bactericidal activity
↑XOR serum level
↑XO/XDH activity
↑XOR expression
(a)
rombosis
Athe ros clerosis
Vascular tone regulation
ROS
Permeabilization
NO
XOR
Endothelial dysfunction
(b)
F : Prophlogistic action of reactive oxygen (ROS) and nitrogen (RNS) species. (a) Interferon and other cytokines increase xanthine
oxidoreductase gene (XOR) expression as well as the conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and XOR serum
level (reviewed in []). XOR-derived ROS and RNS mediate the endothelial and phagocytic cell activation that is functional in antibacterial
defense (reviewed in []). (b) XOR products induce endothelial permeabilization and dysregulation of vascular tone, which may lead to
thrombosis and atherosclerosis (reviewed in []).
products together with the oxidants generated by NAD(P)H
oxidase and NO synthase may also modulate another endo-
thelial cell function, the regulation of arteriolar tone via NO
production, which has local and systemic vasodilating activ-
ity and causes XOR inhibition (Figure (b)). NO is produced
by endothelial NO synthase that is inhibited either by ROS or
under hypoxic conditions. In these circumstances, NO gener-
ation is assured by the nitrite reductase activity of both XOR
and NAD(P)H oxidase, which undergo reciprocal activation
by generating O2∙−(reviewedin[]).Astheactivitiesof
these enzymes are interdependent in the endothelium, the
nal outcome is the result of a physiopathological balance
amongst their activities. us, it is not surprising that both
XOR activity and its inhibition by allopurinol may induce
endothelial dysfunction and promote platelet aggregation, as
well as aggravating hypertension and cardiovascular diseases
[].
In patients with coronary disease, the treatment with
the angiotensin receptor blocker losartan reduced the endo-
thelium-bound XOR activity and XOR inhibition with oxy-
purinol improved endothelium-dependent vasodilation, sug-
gesting that endothelial dysfunction in coronary disease
is at least in part dependent on angiotensin II-dependent
endothelial XOR activation []. In patients with metabolic
syndrome, XOR inhibition by allopurinol reduces myeloper-
oxidase and malondialdehyde blood levels, while increasing
theow-mediateddilation,suggestingthatXOR-induced
oxidative stress contributes to endothelial vasomotor dys-
function[].eunderlyingmechanismissupposedtobe
the reduced bioavailability of NO due to the reaction of NO
with O2∙−(reviewed in []). However, in grade drug-
na¨
ıve hypertensive subjects a dietary nitrate load reduces
systolic and diastolic blood pressure. is eect is related to
an increased NO generation, which is signicantly attenu-
ated by allopurinol and is associated with higher levels of
erythrocytic XOR expression and nitrite reductase activity
in hypertensive patients in comparison to normotensives
volunteers []. e eects of ROS generated by human XOR
on cardiovascular disease have been detailed in two recent
publications (reviewed in [, ]).
XORmayproduceROSandNO,whicharebothrequired
for the formation of normal granulation tissue and wound
healing. In vitro keratinocytes and endothelial cell prolif-
eration and migration were increased by H2O2and nitrite.
XOR expression was upregulated shortly aer wounding at
the wound edge. Locally applied allopurinol, as well as a
tungsten-enriched diet that drastically lowered XOR activity,
signicantly delayed wound healing in mice. e eect was
reversed and angiogenesis improved with the topical H2O2
administration, strongly suggesting that XOR contributes to
wound repair [].
4. Pro- and Antitumorigenic Activity of
Xanthine Oxidoreductase Products
In both experimental and clinical pathology, the level of XOR
expression was oen found to be higher or lower in cancer
tissues compared with the corresponding normal tissue or to
Oxidative Medicine and Cellular Longevity
Promotion
Dierentiation
Transformation
Metastasization
Progression
Angiogenesis Apoptosis
PROS
CONS
CANCER
F : Cancer pathogenesis: ambiguousrole of xanthine oxidore-
ductase (XOR). XOR-derived ROS may activate genes responsible
for each phase of cancer development (reviewed in []) as well as
genes that promote antioncogenic activities (reviewed in []).
the normal tissues bordering cancerous tissues (reviewed in
[]). In particular, XOR expression and activity in neoplastic
human tissues have been recently addressed and discussed
together with the XOR role in dierentiation and oncogenesis
(reviewedin[]).Moreover,XORproductshavebeenasso-
ciated with both the process of oncogenesis ([], reviewed in
[]) and its prevention ([], reviewed in []) (Figure ).
e level of XOR activity was higher than normal and
that of paraoxonase l, a free radical scavenger enzyme, was
lower in the serum of patients with various cancer illnesses
[].Alowactivityofvariousoxidativeenzymes,inparticular
XOR,hasbeenreportedtocorrelatewithcellproliferation
in dierent settings, including cancer, and a hypothesis has
been formulated that a low level of free radicals may stimulate
cancer cell growth []. XOR can also confer a cancer-
promoting action through the above-discussed proinam-
matory activities of ROS and RNS.
e analysis of a vast cohort of women followed for
years showed a dose-dependent risk of breast cancer by
alcohol consumption [] and a mechanism involving XOR-
derived ROS has been proposed for the pathogenesis of this
cancer. XDH is expressed at high levels by mammary epithe-
lium,particularlyinrelationtolactation,andcanproduce
ROS by oxidizing ethanol [] as well as acetaldehyde and
NADH, which are generated by alcohol catabolism. ROS can
be responsible for DNA damage, mutagenesis, and neoplastic
transformation, especially in aged breast tissue with high iron
levels and low antioxidant levels (reviewed in [, ]).
XOR is an upstream regulator of various molecules with
transduction signal functions in dierent pathways, which
may result in either pro- or antitumorigenic signaling.
In human lung microvascular endothelial cells, XOR was
shown to increase the expression of the tumor suppressor
protein p, which is very oen mutated and deactivated in
humancancer.XORinducedoxidativestress,DNAdamage,
and the ROS-dependent upregulation of p protein, with the
consequent activation of the caspase enzymatic cascade and
apoptosis [].
In T-L murine cells, the ROS produced by the NADH-
oxidizing activity of XOR were able to stimulate the acti-
vation of peroxisome proliferator-activated receptor-gamma
(PPAR-𝛾), which belongs to the nuclear hormone receptor
superfamily. is ligand-activated intracellular transcrip-
tion factor has antiproliferative and antioncogenic activities
because it can favor cell dierentiation and inhibit angiogen-
esis [].
XOR-derived ROS can modulate the expression of the
inammation mediator, cyclooxygenase- (COX-), by either
increasing or decreasing its expression. e XOR-dependent
COX- expression in newborn mice was essential for regular
kidney development, and the lack of XOR was associated
with renal hypoplasia and dysplasia []. Additionally, XOR
depletion in primary renal epithelial cells induced positive
immunostaining for mesenchymal cell type markers and the
lack of reactivity to E-cadherin associated with cell morphol-
ogy changes from a cuboidal to myobroblastic shape, which
indicated epithelial to mesenchymal transition []. However,
a high XOR level in human mammar y epithelium lowered the
COX- and matrix metalloprotease expression levels, which
are crucial for cell migratory activity and thus for tumor
progression and ability of metastasis formation [].
XOR-generatedoxidantscanturnonthenuclearfactor
kappa-light-chain-enhancer of activated B cells (NF-𝜅B) in
rat liver both during ischemia [] and in type diabetes [].
NF-𝜅B is a transcription factor that is usually activated during
chronic inammation and in cancer, where it promotes the
production of immunological cytokines and the expression
of a set of antiapoptotic genes.
In U-MG cells, derived from human brain, chemically
induced hypoxia increased XOR activity and the level of
XOR-derived ROS, which upregulated hypoxia-inducible
factor- alpha (HIF-𝛼) []. is transcription factor is over-
expressed in hypoxia and induces angiogenesis, as well as can-
cer invasion, thus contributing to both tumor development
and progression.
5. Conclusions
Mammal XOR is the end product of a complicated evolu-
tionary process leading to a hyperregulated enzyme with low
specicity and highly versatile activity. In mammals, XOR
has acquired many functions through the production of ROS,
NO, and RNS, whereby it is involved in the triggering of
key biological cell pathways and in the regulation of several
physiological and pathological conditions. For these reasons,
XOR represents the two faces of free radicals, which can
have either negative or positive eects. XOR-derived RNS
andROSmayhaveacytotoxiceect.isactivitymaybe
responsiblefortissuedamageinhypoxia/reoxygenationand
ischemia/reperfusion injury. However, this cytotoxic eect
can be pharmacologically exploited to obtain selective can-
cerous cell killing by conjugating XOR to a specic antibody.
Oxidative Medicine and Cellular Longevity
XOR activity increases during infectious diseases and its cyto-
toxic action is useful for the defenses against bacteria. Addi-
tionally, XOR-derived NO and ROS have proinammatory
activity because they regulate endothelial functions, by both
increasing the permeability of vascular lining and modulating
the arteriolar tone. For this reason, XOR has been implicated
in hypertension, cardiovascular diseases, and atherosclerosis.
XOR-derived ROS are also involved in cancer pathogenesis
because they may promote neoplastic transformation by
activating target genes with prophlogistic, antiapoptotic, and
proliferative actions. Moreover, they favor the progression to
malignancy by inducing angiogenesis and cell migration. On
the other hand, XOR products may activate the expression
of the proapoptotic protein p and of transcription factors
belonging to the nuclear hormone receptor superfamily with
antitumorigenic and antiproliferative activity, promoting cell
dierentiation and inhibiting angiogenesis.
Highlights
(i) XOR-derived ROS, NO, and RNS have proinamma-
tory and bactericidal activities.
(ii) XOR products may be cytotoxic in many circum-
stances.
(iii) XOR products modulate endothelial function and
arteriolar tone.
(iv) XOR products may induce mutagenesis, cell prolifer-
ation, and tumor progression.
(v) XOR products are associated with apoptosis and cell
dierentiation.
Abbreviations
COX-: Cyclooxygenase-
FAD: Flavin adenine dinucleotide
HO∙:Hydroxylradical
H2O2: Hydrogen peroxide
Mo-co: Molybdenum-containing molybdopterin
cofactor
NAD+: Oxidized nicotinamide adenine dinucleotide
NADH: Reduced nicotinamide adenine dinucleotide
NF-𝜅B: Nuclear factor kappa-light-chain-enhancer
of activated B cells
NO: Nitric oxide
ONOO−:Peroxynitrite
RNS: Reactive nitrogen species
ROS: Reactive oxygen species
O2:Molecularoxygen
O2∙−:Superoxideanion
XDH: Xanthine dehydrogenase
XO: Xanthine oxidase
XOR: Xanthine oxidoreductase.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgment
is work was supported by the Pallotti Legacies for Cancer
Research.
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