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Part Two: Free Radical Reaction in Biological Systems
The presence of free radicals in biological materials was discovered more than 50 years ago
{Ref: Commoner B, Townsend J, Pake GE, Nature, 1954, 174:689–691}. Soon thereafter,
Denham Harman hypothesized that oxygen radicals may be formed as by-products of
enzymic reactions in vivo, implicating them as the cause of degenerative process of
biological aging {Ref: Harman D, 1956, J. Gerontol 11:298–300}. The subject of free radicals
in biology has since developed into an interdisciplinary study of compelling public interest.
In a recent review of the subject, Dormandy notes that “in living systems, enzymic and free
radical processes are inextricably linked. On the one hand enzymes can both beget and
destroy free radicals; on the other, free radicals can both stimulate and destroy enzymes”
(Ref: Dormandy TL, Ann R Coll Surg Engl.,1980, 62:188-194}.
Enzymes as we all know constitute a perfect homoeostatic system, maintaining a balance
between energy-producing and energy-consuming pathways, most tellingly represented by
interlocking cycles. Based on our experience with chemical systems, we may be tempted to
conclude that free radical generation along normal metabolic pathways would be hugely
unlikely as a biological phenomenon, since such in-vivo free radical activity would inevitably
be non-homoeostatic, non-cyclic, irreversible and wasteful in terms of energy. However,
there is now much accumulated experimental evidence that free radicals, especially
reactive oxygen species (ROS; previously described in Part One) as well as some reactive
nitrogen species (RNS), in particular nitric oxide (·NO) and peroxynitrite (ONOO−), are part
and parcel of aerobic life and actively participate in phagocytosis, inflammation,
and apoptosis.
Several external sources of free radicals and oxidants are known. They include pollutants,
cigarette smoke, radiation, medication (especially drugs such as antibiotics that depend on
quinoid groups or metals for their activity, and antineoplastic agents), etc. Some
endogenous sources of free radicals are mitochondria, phagocytes, cellular oxidase
systems such as NADPH oxidase, xanthine oxidase and peroxidases, enzymatic reactions
associated with prostaglandin synthesis and the cytochrome P450, microsomes,
peroxisomes, exercise, inflammation, etc. {Ref. Halliwell, B. & Gutteridge, J.M.C. (1999):
“Free Radical in Biology and Medicine”, Oxford Science Publications, New York}.
Consider, by way of example, the NADPH oxidase system. The NADPH oxidase complex is a
cluster of plasma membrane–associated enzymes that donate an electron from NADPH to
molecular oxygen to produce superoxide.
2O2 + NADPH 2 O2- + NADP+ + H+
It is a complex enzyme consisting of two membrane-bound components and three
components in the cytosol, plus the signalling proteins Rac1 or Rac2 that are members of
the Rac subfamily of the Rho family of GTPases. Under normal circumstances, the enzyme
complex is latent in neutrophils of the immune system, and is activated to assemble in the
membranes during respiratory burst.
For a long time, superoxide generation by an NADPH oxidase was considered an anomaly
only found in professional phagocytes. In recent years, six homologs of the cytochrome
subunit of the phagocyte NADPH oxidase have been found: NOX1, NOX3, NOX4, NOX5,
DUOX1, and DUOX2. These homologs along with the phagocyte NADPH oxidase itself
(NOX2/gp91phox) are now referred to as the NOX family of NADPH oxidases. These
enzymes share the capacity to transport electrons across the plasma membrane and to
generate superoxide and other downstream reactive oxygen species (ROS). The enzyme
complex has been recognized as an important source of ROS in vascular cells. The
generation of ROS radicals is prompted in response to receptor agonists such as growth
factors or inflammatory cytokines that signal through the Rho-like small GTPases Rac1 or
Rac2.
The pathway by which reactive nitrogen species (RNS) are formed in biological tissues is an
oxidative process in which short-lived nitric oxide (·NO) is derived from the guanidino
nitrogen in the conversion of L-arginine to L-citrulline. This reaction is catalysed by NO
synthase and involves oxygen uptake like the “respiratory burst”. Depending on the
microenvironment, ·NO can be converted to various other reactive nitrogen species such as
nitrosonium cation (NO+), nitroxyl anion (NO−) or peroxynitrite (ONOO−). ·NO is a free
radical signal-transducing agent and is present in a variety of cell types, including vascular
endothelial cells, smooth muscle cells, platelets, neuronal cells, macrophages, and
neutrophils.
Free-radical reactions are intrinsic to a majority of the metabolic and synthetic reactions
carried out by eukaryotic cells and, as such, are required for life. ATP production in
mitochodria, for example, involves the generation of ROS at the cristae of inner
mitochondrial membrane during the final and most important step of cellular respiration,
the electron transport chain (ETC).
Electron transport chain (ETC)
The ETC describes the sequential flow of electrons through an enzymatic series of electron
donors and acceptors via oxidation-reduction reactions, with each acceptor protein along
the chain having a greater reduction potential than the previous. Oxygen acts as the
terminal electron acceptor within the ETC. Passage of electrons between donor and
acceptor releases energy which is used to actively pump protons into the inter-membrane
space from the mitochondrial matrix. This generates a proton electrochemical-potential
gradient across the inner membrane. This proton gradient is discharged with the protons
moving back to the mitochondrial matrix via protein channels called ATP synthase located in
the inner membrane and used to power ATP synthesis from adenosine diphosphate (ADP)
and inorganic phosphate (Pi). A somewhat simplified illustration of ETC is shown below.
In normal conditions in ECT, the oxygen is reduced to produce water. The literature
suggests that anywhere from 2 to 5% of the total oxygen intake during both rest and
exercise is prematurely and incompletely reduced to give the superoxide radical (·O2- ) via
electron escape. Electrons appear to escape from the ETC at the ubiquinone-cytochrome c
level. Superoxide is not particularly reactive by itself, but can inactivate specific enzymes or
initiate lipid peroxidation in its protonated form, hydroperoxyl HO2..
Eicosanoids
Free radicals have been shown to mediate the biosynthesis of a number of important
oxygenated metabolites, among them the ubiquitous eicosanoids which are critical to the
regulatory function of cells. Eicosanoids are not stored within cells, but are synthesized as
required when a cell is activated by mechanical trauma, cytokines, growth factors or other
stimuli. The eicosanoids comprise several compounds, which include prostaglandins,
thromboxanes and leukotrienes; most are produced by the oxidation of arachidonic acid, a
20-carbon polyunsaturated fatty acid (all-cis-5,8,11,14-eicosatetraenoic acid), catalysed by
(Source: Prabha Balaram, personal communication)
PGG2/H2 synthase (sometimes referred to as cyclooxygenase, COX). The structure of
arachidonic acid (20:4, ω-6) is shown below.
Arachidonic acid
Similarly, the oxidation of small molecules by molecular oxygen catalysed by the heme-
containing cytochrome P450 enzyme family is also thought to involve intermediary free
radicals.
Prostaglandins, which are found throughout the body, are known to act like ‘local
hormones’. For example, administration of remarkably small doses of some prostaglandins
stimulates uterine contractions and can cause abortion. Imbalances in prostaglandins can
lead to nausea, diarrhoea, inflammation, pain, fever, menstrual disorders, asthma, ulcers,
hypertension, drowsiness, or blood clots. The most common prostaglandins are PGE1, PGE2,
PGF1α and PGF2α [ PG means prostaglandin, E means the keto alcohol and F means the diol,
the subscript numbers refer to the number of double bonds and α refers to the
configuration of the –OH at carbon 9 (cis to the carboxyl side chain)].
The biological oxidation leading to PGE2 from arachidonic acid, catalysed by COX is
illustrated below. The enzyme contains two active sites: a cyclooxygenase site, where
arachidonic acid is converted into the hydroperoxy endoperoxide prostaglandin G2 (PGG2),
and a heme with peroxidase activity, responsible for the reduction of PGG2 to PGH2. The
reaction proceeds through H atom abstraction from arachidonic acid by a tyrosine radical
generated by the peroxidase active site. Two O2 molecules then react with the
arachidonic acid radical, yielding PGG2.
It has been suggested that the signal molecule ·NO may also initiate prostaglandin synthesis
by reacting with superoxide anion to produce peroxynitrite, which oxidizes the heme iron.
The oxidized heme then accepts an electron from a nearby tyrosine residue (Tyr385).
The resulting tyrosine radical extracts an H atom from arachidonate at the doubly allylic
carbon 13 to yield a carbon-centred radical. Oxygen adds to carbons 9 and 11 and the
cyclopentane ring is formed. Further oxidation leads to the hydroperoxy endoperoxide
intermediate, Prostaglandin G2 (PGG2). This is quickly metabolized by the peroxidase activity
intrinsic to COX to give PGH2. Prostaglandin-H2 E-isomerase catalyzes the conversion of
PGH2 to PGE2, while Endoperoxide reductase converts PGH2 to PGF2α. The latter is also
obtained by the action of the enzyme Prostaglandin-E2 9-reductase on PGE2.
(Source: en.wikipedia.org/wiki/Cyclooxygenase)
Oxidative Stress
When produced in excess, free radicals and oxidants generate a phenomenon called
oxidative stress, a deleterious process that can damage all major cellular constituents and
thus contribute to the pathogenesis of diseases such as cancer, diabetes mellitus,
atherosclerosis, neurodegenerative diseases, rheumatoid arthritis, ischemia/reperfusion
injury and obstructive sleep apnoea. Oxidative stress can arise when cells cannot adequately
destroy the excess of free radicals formed. In other words, oxidative stress results from an
imbalance between formation and neutralization of ROS and RNS.
Redox signalling
At low or moderate concentrations, however, ROS and RNS exert beneficial effects on cellular responses and immune function. Their role as regulatory mediators in signalling processes has been well evidenced {Ref: Wulf Dröge, Physiol Rev, 2002, 82:47-95}.
This process wherein they act as cellular messengers is dubbed redox signalling as the signal
is delivered through redox chemistry. As described by Dröge, “Redox signalling is used by a
wide range of organisms, including bacteria, to induce protective responses against
oxidative damage and to reset the original state of ‘redox homeostasis’ after temporary
exposure to ROS. Many of the ROS-mediated responses actually protect the cells against
oxidative stress and re-establish redox homeostasis. Higher organisms, however, have
evolved the use of ·NO and ROS also as redox signalling molecules for other physiological
functions. These include regulation of vascular tone, monitoring of oxygen tension in the
control of ventilation and erythropoietin production, and signal transduction from
membrane receptors in various physiological processes”.
ROS normally also participate in a number of other important cellular processes besides cell
signalling. These include gene expression, cellular death and senescence, regulation of
growth, oxygen sensing, activation of matrix metalloproteinases, and angiogenesis.
Generation of reactive oxygen species (ROS) by NOX-based NADPH oxidases activate redox-
dependent signalling pathways and contribute to development of oxidative stress in
vascular disease. Aberrant redox signalling can contribute to the pathogenesis of vascular
disease by altering endothelial cell function, enhancing vascular smooth muscle cell growth
and proliferation, stimulating expression of pro-inflammatory genes, and modulating
reconstruction of extracellular matrix. High blood pressure is in part determined by elevated
total peripheral vascular resistance, which is ascribed to dysregulation of vasomotor
function and structural remodelling of blood vessels. {Ref: Lee MY and Griendling KK,
Antioxid Redox Signal. 2008, 10:1045–1059}.
A situation where cells may be induced to produce excessive ROS is that caused by
abnormal environments such as hypoxia or hyperoxia. Yet another is infection by
microorganisms such as viruses and bacteria which causes the immune system to mount in
host defence what is literally a free radical attack on the invading pathogens. The free
radicals are primarily produced by neutrophils which comprise the bulk of the immune
system’s white cells (leukocytes). These engulf and kill the microorganisms
via phagocytosis.
What are these free radicals? Given that neutrophil mitochondria hardly participate in
ATP synthesis and the neutrophils largely depend on glycolysis for their energy, how are
these free radicals formed?
Two types of free radicals are formed in neutrophils. The first is the reactive oxygen
intermediates formed by the activity of nicotinamide adenine dinucleotide phosphate
oxidase (NADPH oxidase), the enzyme of the “respiratory burst”. The second type includes
reactive nitrogen intermediates, the first member of them, nitric oxide being produced by
nitric oxide synthase. Upon activation neutrophils have increased oxygen consumption, a
process known as the respiratory burst. During this stage, oxygen is univalently reduced
by NADPH oxidase to superoxide anion or its protonated form, perhydroxyl radical, which
then is catalytically converted by action of superoxide dismutase to hydrogen peroxide:
O2 + e- + H+ → O2H = O2
- + H+
O2- + O2
- + 2H+ → O2 + H2O2
Hydroxyl radical (·OH), the most reactive free radical in vivo, is formed by several ways,
among which the decomposition of H2O2 catalyzed by Fe2+ is the most important. This
reaction is known as the Fenton reaction.
Fe2+ + H2O2 → Fe3+ + OH- + ·OH
The pathway by which nitric oxide radical (·NO) and other reactive nitrogen species are
formed has been described earlier. Peroxynitrite (ONOO−), a biological oxidant formed from
the reaction of nitric oxide with the superoxide radical, is associated with much pathology,
including neurodegenerative diseases, such as multiple sclerosis. Carbon dioxide catalyses
the isomerization of peroxynitrite to NO3- via an intermediate, presumably ONOOCO2-
which undergoes homolysis to trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*),
transient radicals which are quenched by uric acid. Interestingly, nitroglycerin which over a
century has been used as a drug in the form of tablets, sprays or patches for treating and
preventing attacks of angina pectoris, exerts its effects because of its conversion to ·NO in
the body by mitochondrial aldehyde dehydrogenase. Nitric oxide is a natural vasodilator in
in the body.
Enzymic Antioxidants
Paradoxically, the major hint of the wide occurrence of free radical activity in the body
came from the observation of widespread distribution of superoxide dismutase (SOD)
family of enzymes in cells {Ref: McCord JM and Fridovich I, J Biol Chem, 1969, 244:6049–
6055}. These enzymes are powerful free radical scavengers, and it has been shown that they
particularly target the superoxide-ion free radical, O2- , in the aqueous phase. SODs are
metal-containing enzymes that depend on the bound manganese, copper or zinc for their
antioxidant activity. In mammals, the manganese-containing enzyme is most abundant in
mitochondria, while the zinc or copper forms are predominant in cytoplasm. SODs catalyze
the conversion of the two superoxide-ion radicals into the peroxide ion radical and oxygen.
O2- + O2
- → O2 2- + O2
SODs also feature in protective mechanisms against light-induced ROS formed during
photosynthesis either by electron-transfer from excited chlorophyll molecules to oxygen or
by transfer of electrons to oxygen from carriers such as ferredoxin. This protective
mechanism adopted by plants is additional to the dissipation as heat the excess light energy
absorbed by pigments.
Eukaryotic cells possess besides SODs two other important enzymatic oxidants against free
radicals:
Catalase - this degrades hydrogen peroxide to water and oxygen, and hence completes the
detoxification reaction started by SOD, and
Glutathione peroxidase – these selenium-containing enzymes degrade hydrogen peroxide
and reduce organic (lipid) peroxides to alcohols.
Antioxidant properties of Uric acid
In humans, uric acid is considered a major antioxidant that may protect against aging and
oxidative stress. It is the most abundant antioxidant in plasma (normal levels are in the
range 3.5 to 7.2 mg/dl) and is the end product of purine degradation. Xanthine, a product
on the pathway of purine degradation, is catalytically oxidised to uric acid by
xanthine oxidase, an enzyme that generates ROS. Uric acid's antioxidant activities are
complex, given that it does not react with some oxidants, such as superoxide, but does act
against peroxynitrite, peroxides, and hypochlorous acid {Ref: http://en.wikipedia.org/ wiki/
Antioxidant}.
It was recently shown that uric acid activates NADPH oxidase resulting in increased
production of ROS, leading to decreased bioavailabilty of ·NO and increased protein
nitration {Ref: Sautin Y, Nakagawa T, Zharikov S and Johnson RJ, Am J Physiol Cell, 2007,
293: C584-C596}. Uric acid has been shown also to react directly and irreversibly with ·NO
resulting in the formation of 6-aminouracil and depletion of ·NO, which is a potent
vasodilator. The reaction proceeds even in the presence of oxidants peroxynitrite and
hydrogen peroxide and is at least partially blocked by glutathione. Thus under conditions of
oxidative stress in which uric acid is elevated and intracellular glutathione lessened, the
depletion of ·NO levels leads to endothelial dysfunction {Ref: Gersch C et al. Nucleosides
Nucleotides Nucleic Acids, 2008, 27: 967-978}. The endothelium plays a major role in
maintaining vascular tone and modulating blood flow and pressure, and its dysfunction is
identified as contributing to hypertension, arterial stiffness and cardiovascular diseases.
One interesting fact that has emerged from recent studies is that high consumption of
sugars containing fructose can raise uric acid concentrations in the blood. It has been
proposed that fructose-induced hyperuricemia may play a major role in the development of
hypertension, obesity, and metabolic syndrome (a name for a group of risk factors that
occur together and increase the risk for coronary artery disease, stroke, and type 2-
diabetes) and in the subsequent development of kidney disease {Ref: Johnson RJ et al, Am J
Physiol Cell, 2007, 86: 899-906}.
Lipid Peroxidation
One of the best known toxic effects of oxygen radicals is damage to cellular membranes
(plasma, mitochondrial and endomembrane systems), which is initiated by a process known
as lipid peroxidation. A common target for peroxidation is unsaturated fatty acids present
in membrane phospholipids {Ref:www.vivo.colostate.edu/hbooks/pathphys/misc_topics/
radicals.html}.
Lipid peroxidation is a self-propagating phenomenon terminated by antioxidants (see below; R•=free radical species (ROS, RNS), L=lipid, A=antioxidant).
1. Initiation: R• + LH → RH + L•
2. Propagation: L• + O2 → LOO•
LOO• + LH → LOOH + L•
3. Termination: L• + AH → LH + A•
A• + LOO• → LOO-A
Nitric oxide and its oxidant metabolites (e.g. ONOO− ) can both stimulate and inhibit lipid
peroxidation, depending on relative concentrations of ·NO, ROS, and antioxidants, with all
interactions in turn being influenced by the aqueous-lipid solubility and relative rates of
reaction of the participating reactive species {Ref: Bloodsworth A, O’Donnell VB and
Freeman BA, Arteriosclerosis, Thrombosis, and Vascular Biology.2000; 20: 1707-1715}. Thus,
lipid peroxidation is inhibited by ·NO when its concentration exceeds that of superoxide
radical; the result is the termination of lipid radical–mediated chain propagation reactions
(i.e. ROO· + ·NO→ROONO). Likewise, the generation of metal-nitrosyl derivatives by ·NO
quenches the initiation of lipid oxidation by metals. For example, myoglobin and
hemoglobin oxoferryl free radical species (·Mb-FeIV=O/·Hb-4FeIV=O) are reduced to their
respective ferric (met) forms on reaction with ·NO, thus affording protection against
oxidative damage by oxoferryl Mb/Hb. Additionally, methemoglobin (metHb) (FeIII; does
not bind oxygen) binds ·NO to form a nitrosyl-hemoglobin (·NO-Hb) intermediate that loses
its ability to oxidize linoleic acid and produce conjugated dienes as well as the ability to co-
oxidize substrates such as β-carotene. In general, when ·NO complexes with
metalloproteins, lipids are protected from further oxidation by metals and oxidant
metabolites of ·NO. Nitric oxide also inhibits the oxidation of LDL by scavenging LOO· via
chain-terminating interactions of ·NO and other reactive nitrogen species, yielding oxidized
nitrogen-containing lipid products.
Peroxidation of membrane lipids can lead to altered permeability, altered activity of
membrane receptors and even decreased activity of membrane-bound enzymes such as
sodium pumps. Products of lipid peroxidation, for example, malondialdehyde, irreversibly
disrupt enzymes, receptors, and membrane transport mechanisms. In acute ischaemic
stroke, in vivo concentrations of lipid peroxidation products are significantly increased,
arising from excess free radical activity. Plasma concentrations of cholesteryl ester
hydroperoxides (CEOOH) are sensitive and specific markers of lipid peroxidation, and
correlate positively with infarct volume, calculated by computed tomography, and clinical
severity, determined by the National Institute of Health Stroke Scale {Ref: Waring WS, QJM
(2002), 95: 691-693}. This emphasizes the role of oxidative stress in mediating cerebral
ischaemic tissue damage.
As with lipids, proteins and DNA also suffer damage by free radicals; the breakdown
patterns are as shown below.
Apoptosis
If too much damage is caused to its mitochondria, a cell undergoes apoptosis or
programmed cell death. Cell suicide, or apoptosis, is the body's way of controlling cell death
and involves free radicals and redox signalling. This self-destructing act is a highly
orchestrated one and involves the release of cytochrome c from the mitochondria, which
along with another protein factor, Apaf-1, activates caspase-9 and initiates the cell death
cascade. The cascade is controlled by pro- and anti-apoptotic B-cell lymphoma protein
family members, the most important of them being such as Bcl-2 and Bax. A change in the
balance between these factors can lead to either premature cell death or to unchecked cell
division. The caspase-9 cleaves the proteins of the mitochondrial membrane, causing it to
break down and start a chain reaction of DNA fragmentation, protein denaturation and
formation of apoptotic bodies, and eventually phagocytosis of the cell.
Vitamins and other endogenous non-enzymic antioxidants
Understandably, neutrophils have to contain large reserves of endogenous antioxidants
such as glutathione and vitamins C and E. Their ability to maintain these antioxidants in the
reduced state during phagocytosis may prevent death from oxidative suicide.
Glutathione may well be the most important intracellular defence against damage by
reactive oxygen species. It is a tripeptide (glutamyl-cysteinyl-glycine). The cysteine provides
an exposed free sulphydryl group (SH) that is very reactive, providing an abundant target for
radical attack. Reaction with radicals oxidizes glutathione, but the reduced form is
regenerated in a redox cycle involving glutathione reductase and the electron acceptor
NADPH.
Vitamin E , the most biologically active form of which is alpha-tocopherol, is the major lipid-
soluble antioxidant, and plays a vital role in protecting membranes from oxidative damage.
Its primary activity is to trap peroxy radicals in cellular membranes. In the process, it
transiently becomes a radical but is regenerated through the activity of the antioxidants
vitamin C and glutathione.
Vitamin C (Ascorbic acid) can reduce radicals from a wide variety of sources. It does this by
losing an electron to a free radical and remaining stable itself by passing its unstable
electron around the antioxidant molecule.
The powers of antioxidants to limit damage to biological structures have led to the
hypotheses that large amounts of antioxidants might lessen the radical damage causing
chronic diseases, and even radical damage responsible for aging. DNA cross-linking, for
example, engendered by free radical-induced chain reaction involving base pairs in a strand
of DNA, can lead to various effects of aging, especially cancer. Other crosslinking can occur
between fat and protein molecules and leads to wrinkles. Free radicals can oxidize low-
density lipoproteins (LDL), and this is a key event in the formation of plaque in arteries,
leading to heart disease and stroke. There is growing evidence that aging involves, apart
from radical-mediated oxidative damage, progressive changes in free radical-mediated
regulatory processes that result in altered gene expression. However, the enduring interest
in antioxidants as a means to lessen the degenerative causes of biological aging continues.
Nutraceuticals
Presently there is much focus on the possible protective value of a wide variety of plant-
derived antioxidant compounds, particularly those from fruits and vegetables. These include
carotenoids, tocopherols, ascorbates, alpha-lipoic acids, polyphenols and the minerals
copper, zinc and selenium which are natural antioxidants with free radical scavenging
activity. The antioxidant compounds derived from natural products also belong to a new
class of compounds labelled in the marketplace as nutraceuticals. Many have gained
popularity on account of their demonstrated health benefits, among them, flavonoid
polyphenols like epigallocatechin 3-gallate (EGCG) from green tea and quercetin from
apples; non-flavonoid polyphenols such as curcumin from tumeric and resveratrol from
grapes; phenolic acids or phenolic diterpenes such as rosmarinic acid or carnosic acid,
respectively, both from rosemary; and organosulphur compounds including alpha-lipoic
acid and the isothiocyanate L-sulphoraphane, from broccoli and the thiosulphonate allicin,
from garlic {Ref: Kelsey NA, Wilkins HM and Linseman DA, Molecules (2010), 15: 792-814}.
Nonetheless, some recent studies tend to show that antioxidant therapy has no effect on
aging and can even increase mortality brought on by a decrease of normal biological
response to free radicals that creates in its wake a more sensitive environment to oxidation
{Ref: en.wikipedia.org/wiki/Free radical theory_ of_ aging}.
vg kumar das (24 August 2012)
vgkdasorig@gmail.com
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