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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright 2015 by The American Society for Pharmacology and Experimental Therapeutics
Minireview
Arsenic, Reactive Oxygen, and Endothelial Dysfunction
David C. Ellinsworth
Bristol Heart Institute, University of Bristol, Bristol, United Kingdom
Received January 29, 2015; accepted March 17, 2015
ABSTRACT
Human exposure to drinking water contaminated with ar-
senic is a serious global health concern and predisposes
to cardiovascular disease states, such as hypertension,
atherosclerosis, and microvascular disease. The most sen-
sitive target of arsenic toxicity in the vasculature is the
endothelium, and incubation of these cells with low concen-
trations of arsenite, a naturally occurring and highly toxic
inorganic form of arsenic, rapidly induces reactive oxygen
species (ROS) formation via activation of a specific NADPH
oxidase (Nox2). Arsenite also induces ROS accumulation in
vascular smooth muscle cells, but this is relatively delayed
because, depending on the vessel from which they originate,
these cells often lack Nox2 and/or its essential regulatory
cytosolic subunits. The net effect of such activity is at-
tenuation of endothelium-dependent conduit artery dilation via
Introduction
Arsenic is a toxic metalloid present as the 20th most
abundant element in the earths crust (Woolson et al., 1977),
and is a major health concern for .200 million people
worldwide (Naujokas et al., 2013). The Agency for Toxic
Substances and Disease Registry ranks arsenic as number
one on their Priority List of Hazardous Substances (http://
www.atsdr.cdc.gov/spl/), and it is also classified as a Group I
carcinogen by the International Agency for Research on
Cancer (Straif et al., 2009). Human exposure to arsenic can
occur through various industrial processes, as well as
contaminated food and polluted air. However, the highest
risk source of intoxication is contaminated drinking water.
Countries that are affected by arsenic-contaminated drinking
water include Bangladesh, India, China, Taiwan, Mongolia,
Mexico, Argentina, Chile, and specific regions of the United
States (Argos et al., 2010; Rodriguez-Lado et al., 2013).
Indeed, the serious risk to 3577 million people in Bangladesh
dx.doi.org/10.1124/jpet.115.223289.
ABBREVIATIONS: As2O3, arsenic trioxide; AsV, arsenate; AsIII, arsenite; CAD, coronary artery disease; DHE, dihydroethidium; DMAIII,
dimethylarsinous acid; EC, endothelial cell; EDH, endothelium-derived hyperpolarization; eNOS, endothelial nitric oxide synthase; H2O2, hydrogen
peroxide; LSEC, liver sinusoidal endothelial cell; MMAIII, monomethylarsonous acid; Nox, NADPH oxidase; NO, nitric oxide; NOS, nitric oxide
synthase; O22, superoxide; ONOO2, peroxynitrite; ROS, reactive oxygen species; S1PR1, shingosine-1-phosphate receptor 1; SOD, superoxide
dismutase; VSMC, vascular smooth muscle cell.
458
http://dx.doi.org/10.1124/jpet.115.223289
J Pharmacol Exp Ther 353:458464, June 2015
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superoxide anionmediated scavenging of nitric oxide (NO)
and inhibition and downregulation of endothelial NO synthase,
events that are temporally matched to the accumulation of
oxidants across the vessel wall. By contrast, ROS induced by
the more toxic organic trivalent arsenic metabolites (mono-
methylarsonous and dimethylarsinous acids) may originate
from sources other than Nox2. As such, the mechanisms
through which vascular oxidative stress develops in vivo under
continuous exposure to all three of these potent arsenicals are
unknown. This review is a comprehensive analysis of the
mechanisms that mediate arsenic effects associated with
Nox2 activation, ROS activity, and endothelial dysfunction, and
also considers future avenues of research into what is
a relatively poorly understood topic with major implications
for human health.
alone has been described by the World Health Organization as
the largest poisoning of a population in history (Argos et al.,
2010), and the current estimate of 20 million for China is
likely an underestimate of the actual affected population due
to the millions of wells as sources of drinking water that are
still to be tested for contamination, with the expectation that
such testing will take several decades to complete (Rodriguez-
Lado et al., 2013). Numerous epidemiologic studies have
provided evidence that chronic arsenic intoxication predis-
poses to a number of cardiovascular diseases, including
hypertension, coronary artery disease (CAD), stroke (carotid
atherosclerosis), and microvascular abnormalities (arterio-
sclerosis and Blackfoot disease), as well as diabetes mellitus,
skin lesions, neurodegenerative disorders, and cancer (pri-
marily of the lung, bladder, kidney, liver, and skin) (for
review, see Chen, 2014).
In contaminated water, arsenic is present in the inorganic
forms of pentavalent arsenate (AsV) and trivalent arsenite
(AsIII) (Vahter, 2002). Once ingested, AsV is rapidly reduced
(primarily in the blood) to the more reactive and toxic AsIII. This
is then methylated (primarily in the liver) to monomethylarsonic

acid, with subsequent reduction/methylation cycles producing
monomethylarsonous (MMAIII), dimethylarsinic, and dimethyl-
arsinous (DMAIII) acids (Vahter, 2002). With the exception
of AsV reduction to AsIII, which is catalyzed by purine nu-
cleoside phosphorylase, these steps are catalyzed by glu-
tathione S-transferases v1 and v2 (using glutathione as
the reducing agent) and arsenic methyltransferase (using
S-adenosylmethionine as the methyl donor) (De Chaudhuri
et al., 2008) (Fig. 1). Although the methylated species are the
primary products excreted in urine, their trivalent forms
(i.e., MMAIII and DMAIII) are intrinsically more toxic than
AsIII, with the pentavalent species themselves being gener-
ally nontoxic at experimental levels relevant to human
environmental exposure (Aposhian et al., 2003). Notably,
arsenic trioxide (As2O3) is used clinically as a highly effective
treatment of acute promyelocytic leukemia and is converted
to AsIII and, subsequently, to methylated species in vivo
after administration (Chen et al., 2013).
In the vascular system, and primarily in endothelial cells
(ECs), exposure to arsenic induces overproduction of reactive
oxygen species (ROS), which modulate signaling events that
are central to the ability of this cell monolayer to regulate the
tone of the underlying vascular smooth muscle cells (VSMCs),
an event widely designated as endothelial dysfunction
(Bilszta et al., 2006; Verma et al., 2010; Edwards et al.,
2013). Notably, in individuals exposed chronically to arsenic
in drinking water there is a positive correlation between blood
levels of arsenic and plasma markers of ROS accumulation
(Wu et al., 2001). Endothelial dysfunction is widely accepted
as an initiating precursor to the development of chronic
vascular abnormalities (Vanhoutte, 2009), and so elucidation
of the mechanisms that underlie arsenic-induced distur-
bances in the regulation of vascular tone could therefore
provide insight into the associated etiology of hypertension
and vascular disease.
Reactive Oxygen and the Endothelium
In both health and disease, ROS play an important role in
numerous signaling processes in most cell types. Vascular
cells are capable of producing ROS via a number of different
sources, including several NADPH oxidase (Nox) isoforms (see
Fig. 1. Mechanisms of arsenic metabolism. As V ingested through
drinking water is reduced in the blood to trivalent AsIII by purine
nucleoside phosphorylase, which is then methylated in the liver by arsenic
methyltransferase to pentavalent monomethylarsonic acid (MMAV). Sub-
sequent reduction [herein catalyzed by glutathione (GSH) S-transferases
v1 and v2] and methylation cycles lead to production of MMAIII,
dimethylarsinic (DMAV), and DMAIII acids.
Arsenic and Endothelial Dysfunction 459
below), nitric oxide (NO) synthases (NOS), the mitochondrial
electron transport chain, cytochrome P450 epoxygenases,
xanthine oxidase, cyclooxygenases (cyclooxygenase-1 and
-2), and lipooxygenases (Weaver et al., 2012). With the
exception of the Nox family, these sources reduce molecular
O2 to produce superoxide anions (O22) as a byproduct of their
metabolic activity, whereas Nox isoforms synthesize ROS
[variably O22 or hydrogen peroxide (H2O2); see below] as
their sole product. Depending on the origin of synthesis and/or
activity, O22 is further reduced by one of potentially three
superoxide dismutases (SODs) (cytosolic Cu/Zn-SOD, mito-
chondrial Mn-SOD, or extracellular SOD) to produce H2O2
(Zinkevich and Gutterman, 2011), a weaker, but more stable
oxidant that is known to play an important role in vascular
tone regulation in a number of artery types (for examples, see
Matoba et al., 2003; Hatoum et al., 2005; Phillips et al., 2007;
Larsen et al., 2009; Liu et al., 2011b).
Signaling via Nox-derived ROS is integral to a number of
aspects of vascular physiology, such as differentiation,
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angiogenesis, cell survival, growth, and contraction, but
acute and chronic increases in the activity and expression
of Nox isoforms are implicated in the development of
hypertension, atherosclerosis, ischemia-reperfusion injury,
and endothelial dysfunction (Lassegue et al., 2012). The
Nox family consists of seven catalytic subunits, designated
as Nox15 and the dual oxidases Duox1 and Duox2, of
which Nox1, 2, 4, and 5 are expressed in arteries (Lassegue
et al., 2012). With the exception of Nox5, these catalytic
subunits are bound to p22phox as a membrane-associated
heterodimeric complex (designated as cytochrome b558, for
Nox2-p22phox). With the exception of Nox4, which is
constitutively active, the translocation of regulatory cyto-
solic subunits, variably including p47phox, Noxo1, p67phox,
Noxa1, p40phox, and the small GTPase Rac1, to the
plasmalemma and their association with the Nox-p22phox
heterodimer is an essential step to initiate oxidase activity
(Lassegue et al., 2012).
In general, Nox1 is expressed at relatively low levels in ECs
[notable exceptions are certain cultured ECs, including those
obtained from bovine aorta and rat basilar artery, in which it
is highly expressed (Ago et al., 2005)], whereas Nox2 and
Nox4 are much more common in the endothelium, with Nox4
generally being the most abundant (Lassegue et al., 2012).
Specialized roles for Nox2 and Nox4 are believed to rest on
their specific sub-EC distributions (plasmalemma and endo-
plasmic reticulum membranes, respectively; see table 1 in
Lassegue et al., 2012) as well as the ability of Nox4 to produce
H2O2 directly without intermediary O22 (Montezano et al.,
2011; Lassegue et al., 2012).
A number of risk factors for cardiovascular disease are
associated with increased vascular ROS generation that
exceeds endogenous antioxidant capacity (designated as
oxidative stress) (States et al., 2009; Tousoulis et al.,
2011). Indeed, O22 reacts rapidly with endothelium-derived
NO to form the toxic reactive nitrogen species peroxynitrite
(ONOO 2 ), thereby reducing the bioavailability of NO.
Furthermore, ONOO2 exacerbates oxidative stress and
impaired NO by oxidizing the essential endothelial NOS
(eNOS) cofactor (6R)-5,6,7,8-tetrahydrobiopterin, leading to
the uncoupling of eNOS and an increase in O22 generation by
the oxygenase component of the enzyme (Forstermann and
Munzel, 2006).
460 Ellinsworth
Arsenic and NO: Role of Oxidative Stress
A number of studies of isolated vascular cells and tissues
have provided clear evidence that AsIII stimulates the
production of ROS and promotes oxidative stress. In ECs,
the accumulation of both O22 and H2O2 is detectable
within minutes of exposure to low, noncytotoxic, and environ-
mentally relevant concentrations of AsIII (2.55 mmol/l)
(Barchowsky et al., 1999; Straub et al., 2008) (Fig. 2). A
similarly rapid induction of ROS has also been observed in
human bladder epithelial cells exposed to 1 or 10 mmol/l AsIII
(Eblin et al., 2006) and in human keratinocytes exposed to 3
mmol/l AsIII (Cooper et al., 2009).
By contrast, in human aortic VSMCs exposed to 110 mmol/l
AsIII, ROS production takes up to 60 minutes to initiate and
oxidative stress develops over a period of hours (Lynn et al.,
2000). Even at extremely high concentrations (100 mmol/l;
i.e., 1020-fold higher than the generally accepted maxi-
mum level relevant to human environmental/clinical expo-
sure) incubation with AsIII for 30 minutes fails to increase
dihydroethidium (DHE) fluorescence (a specific O22 indica-
tor) in either the media or adventitia of rabbit aortic and iliac
artery segments, although under similar conditions the
metalloid induces an expected robust increase in fluorescence
in aortic valve endothelium (Edwards et al., 2013). Further-
more, in reactivity studies of these vessels incubation with
AsIII for either 30 or 90 minutes does not affect endothelium-
dependent, NO-mediated relaxations to the G proteincoupled
receptor agonist acetylcholine, or to cyclopiazonic acid, an
agent that promotes store-operated extracellular Ca21 influx
by inhibiting the endothelial sarcoendoplasmic reticulum
Fig. 2. Effects of acute and chronic AsIII exposure on endothelium-
dependent, NO-mediated vasodilation. Acute exposure to AsIII reduces
basal NO but does not affect NO-dependent vasodilation, whereas chronic
exposure impairs NO-mediated vasodilation in association with EC and
VSMC O 2 2 accumulation, as well as inhibition of eNOS through
modulation of phosphorylation status (increased Thr497 phosphorylation
and decreased Ser1179 phosphorylation, for bovine eNOS). BH4, tetrahy-
drobiopterin; PP-1, protein phosphatase-1.
Ca21-ATPase (Edwards et al., 2013). It could be argued that
the ability of AsIII at concentrations as low as 0.5 mmol/l to
rapidly induce endothelial nitrotyrosine formation (indicative
of ONOO2 accumulation) (Bunderson et al., 2002; Straub
et al., 2008) implies that O22 accumulates sufficiently to
impair NO bioavailability. However, the aforementioned
findings in rabbit arteries nevertheless suggest that
endothelium-selective increases in ROS formation that follow
acute exposure to AsIII are insufficient to affect the increased
flux of NO associated with vessel relaxation (Edwards et al.,
2013) (Fig. 2). This idea is consistent with the demonstration
that exposure of rats to a single high intravenous dose
(6 mg/kg) of AsIII for 4 hours does not affect acetylcholine-
induced relaxation of ex vivo preparations of aorta (mediated
entirely by NO in this vessel), but does potentiate the
development of tone induced by phenylephrine such that, in
contrast to control preparations, tone is not further enhanced
when NOS activity is inhibited, implying that only basal
levels of NO are affected (Bilszta et al., 2006).
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The generality of these findings relate to observations that
1) acetylcholine-induced relaxation is maintained in aortic
tissue isolated from transgenic mice with endothelium-
selective overexpression of Nox2 (Bendall et al., 2007), and
2) the well known ability of angiotensin II to suppress NO-
mediated relaxation is associated with rapid and global
increases in vascular Nox activity (nonselectively via Nox1
or Nox2, whichever is present) in the intimal, medial, and
adventitial layers (Landmesser et al., 2002; Rey et al., 2002;
Lassegue and Griendling, 2010). As such, suppression of
endothelium-dependent relaxation induced by chronic AsIII
exposure is more likely to be mediated by oxidant accumula-
tion in multiple layers of the vessel wall (see below and Fig. 2).
Consistent with this proposal, more prolonged exposure of
rats to AsIII (1.5 mg/kg per day for 2 weeks) attenuates ex vivo
aortic relaxations to acetylcholine in association with elevated
serum markers of oxidative stress (Kaur et al., 2010). Indeed,
in this particular experimental model of arsenic toxicity,
serum oxidant markers and impaired vessel relaxation (see
Fig. 2) are restored by the peroxisome proliferator-activated
receptor g ligands fenofibrate and roziglitazone and also by
atorvastatin (Jindal et al., 2008; Kaur et al., 2010; Verma
et al., 2010), agents that are associated variously with
increased (6R)-5,6,7,8-tetrahydrobiopterin synthesis (through
the upregulation of GTP-cyclohydrolase-I), reduced serum
levels of asymmetric dimethylarginine (an endogenous NOS
inhibitor), increased eNOS phosphorylation, and suppressed
vascular p22phox expression, nitrotyrosine formation, and
O22 content (Okayasu et al., 2008; Potenza et al., 2009;
Antoniades et al., 2011; Liu et al., 2011a). Time- and
concentration-dependent effects are also apparent after
exposure of rats to environmentally relevant low-to-
moderate AsIII levels in drinking water [20 mg/l; with 10 mg/l
deemed as the safe level by the World Health Organization
(http://www.who.int/water_sanitation_health/publications/2011/
dwq_guidelines/en/)], where acetylcholine-induced aortic re-
laxation is maintained after 2 months but reduced after 7
months exposure (Cifuentes et al., 2009).
AsIII-induced disturbances in endothelial function may not,
however, be simply due to reductions in the bioavailability of
preformed NO and may also involve chronic effects on eNOS
activity and protein expression. For example, in isolated
segments of rat aorta incubated for 14 hours with high

concentrations of AsIII (2550 mmol/l) the attenuation of
acetycholine-induced relaxation is associated with the re-
duced conversion of L-arginine to L-citrulline (suggestive of
reduced NO production by eNOS) (Lee et al., 2003). However,
these authors demonstrate that AsIII-induced attenuation of
eNOS activity is independent of oxidative stress (Lee et al.,
2003), which contrasts with findings in bovine aortic ECs that
a similar high concentration of AsIII (30 mmol/l) suppresses
eNOS activity via ROS-mediated inhibition of protein
phosphatase-1, resulting in derestricted phosphorylation of
eNOS-Thr497 (Seo et al., 2014) (Fig. 2). The reason for these
disparate findings may rest on the duration of AsIII exposure,
i.e., 14 hours (Lee et al., 2003) versus 4 hours (Seo et al., 2014).
Notably, more prolonged (.24 hours) exposure of bovine
aortic ECs to 30 mmol/l AsIII promotes endothelial dysfunction
via coordinated increased Thr497 (equivalent to Thr495 in
human eNOS) phosphorylation, reduced Ser1179 (Ser1177 in
human eNOS) phosphorylation (Fig. 2), and downregulation
of eNOS (Seo et al., 2014).
However, although exposure of rats chronically to drinking
water containing extremely high concentrations (100 mg/l) of
AsIII modulates aortic eNOS phosphorylation and expression
(Kesavan et al., 2014; Sarath et al., 2014), it is currently
unclear whether environmentally relevant levels of AsIII
evoke similar effects in vivo. Indeed, in human aortic ECs
treated with 100 mg/l As2O3 (equating to 0.5 mmol/l)
reductions in NO bioavailability (observed after 1-hour
incubation) are followed by decreased activity and down-
regulation of eNOS (after 72-hour exposure), but although the
authors of this study investigated the significance of these
findings in rats in vivo, neither serum NO metabolites nor
aortic eNOS content were affected (Chou et al., 2007). It
should also be noted that low level MMAIII (100500 nmol/l)
potentiates vasoconstrictor responses to agonists via an
endothelium-independent mechanism mediated by Ca21
sensitization and increased myosin light chain phosphoryla-
tion (Lim et al., 2011).
Arsenic and Nox2
The possibility that AsIII induces EC oxidative stress via
a mechanism involving a Nox-based oxidase was first alluded
to in studies of cultured porcine aortic ECs (Barchowsky et al.,
1999; Smith et al., 2001). Treatment of porcine aortic ECs
with 5 or 10 mmol/l AsIII rapidly induces translocation of Rac1
to membrane fractions and triggers membrane-associated
oxidase activity (Fig. 3), whereas direct treatment of isolated
membranes or cells deficient in p67phox (an indispensable
cytosolic subunit of Nox2) or Rac1 is without effect (Smith
et al., 2001).
In murine liver, sinusoidal ECs (LSECs) AsIII (2.5 or
5 mmol/l for 30 minutes)-induced DHE fluorescence is pre-
vented by gp91ds-tat (Straub et al., 2008) [also known as
Nox2ds-tat (Lassegue et al., 2012)], a synthetic docking
sequence peptide that prevents p47phox from binding to
Nox2 (Rey et al., 2001). AsIII-induced pathophysiological
remodeling of LSECs (defenestration/capillarization and
platelet/endothelial cell adhesion molecule-1 expression) is
also attenuated variously by Nox2ds-tat, genetic deletion of
p47phox and selective pharmacological inhibition of Rac1
(Straub et al., 2008). Furthermore, studies of human
microvascular ECs and LSECs have revealed that AsIII
Arsenic and Endothelial Dysfunction 461
Fig. 3. Mechanisms of AsIII-induced activation of Nox2 in vascular EC.
Exposure to AsIII leads to rapid activation of Nox2 by evoking either the
pertussis toxin (PTx)sensitive translocation of the essential GTPase Rac1
to membrane fractions or through stimulation of another small GTPase,
Cdc42, which induces actin filament reorganization to promote the
translocation of regulatory cytosolic subunits and increase oxidase
activity. The ability of AsIII-induced, Nox2-derived superoxide anions
(O22) to stimulate O22 synthesis/release by mitochondria is not yet clear,
and MMAIII and DMAIII may trigger O22 formation/release from the
distinct sources of mitochondria and the endoplasmic reticulum (ER),
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respectively.
activates a pertussis toxin-sensitive G protein (i.e., Gi)coupled
pathway to induce Rac1 translocation (Straub et al., 2009)
(Fig. 3). Indeed, AsIII-induced angiogenic gene expression in
human microvascular ECs is prevented by pharmacological
blockade or siRNA knockdown of sphingosine-1-phosphate
type 1 receptors (S1PR1) (Straub et al., 2009), and blockade
of S1PR1s prevents both S1P- and AsIII-induced ROS
formation in LSECs (Straub et al., 2009). Pertussis toxin-
sensitive AsIII-induced differentiation of human mesenchy-
mal stem cells, however, is unaffected by blockade of S1PR1s,
but is prevented in a cumulative fashion by selective
blockers of endothelin-1 type A and B receptors (Klei et al.,
2013). Speculatively, AsIII-induced Nox2 activation and
oxidative stress in these ECs may therefore reflect a general
shift toward Gi-coupled cell signaling.
By contrast, exposure of murine saphenous vein ECs to 10
mmol/l AsIII does not directly induce Rac1 translocation, but
instead activates another related small Rho GTPase, Cdc42
(Fig. 3), which, in turn, promotes actin filament reorganiza-
tion, serine phosphorylation of p47phox, and translocation of
p67phox, events that are temporally matched to the rapid
(within 3 minutes) accumulation of O22 in these cells (Qian
et al., 2005). The reasons underlying such differences in
GTPase induction are not yet clear, but are not specific to the
concentration of AsIII applied (Smith et al., 2001; Qian et al.,
2005) and are therefore more likely a reflection of cell-specific
response to the metalloid.
In VSMCs AsIII induces ROS accumulation in a manner
that is prevented by knockdown of p22phox, but observations
that the elevation in ROS is potentiated by supplementation
with NADH, but not NADPH, allude to the activation of an
NADH oxidase but not Nox (Lynn et al., 2000). Indeed, these
particular VSMCs [derived from human aorta (Lynn et al.,
2000)], in contrast to those derived from human resistance
arteries, do not express Nox2 (Touyz et al., 2002), and the
relative delay (up to 60 minutes) in ROS formation induced by
AsIII appears to be more temporally matched to the upregu-
lation of p22phox (Lynn et al., 2000) (Fig. 3). Notably, Nox2 is
barely detectable in VSMCs derived from rat aorta (Lassegue
et al., 2001), a vessel that is frequently used as a model system
for acute and chronic arsenic toxicity in vitro and in vivo (see
462 Ellinsworth
Arsenic and NO: Role of Oxidative Stress). Furthermore,
VSMCs do not consistently express p40phox or p67phox (Touyz
et al., 2002; Cifuentes and Pagano, 2003; Schiffrin and Touyz,
2003), and so the catalytic cytochrome b558 and regulatory
subunits that characterize the Nox2 complex are not
universally present in VSMCs.
Despite compelling evidence that AsIII acutely activates
Nox2, studies of human hepatocytes have shown that
exposure to AsIII at concentrations (under 5 mmol/l) known
to activate this oxidase in ECs (Straub et al., 2008) induces
ROS formation in a manner that is prevented by either the
flavoprotein inhibitor diphenylene iodonium or the mitochon-
drial electron transfer inhibitor rotenone (Li et al., 2014).
Indeed, colocalization studies using DHE and MitoTracker
appear to confirm mitochondria as the principal effector
source of ROS under these conditions (Li et al., 2014).
However, observations that AsIII fails to induce ROS release
from isolated mitochondria (Naranmandura et al., 2011)
would appear consistent with the proposal that AsIII-induced
mitochondrial ROS formation in hepatocytes occurs second-
arily to the activation of Nox2 (Li et al., 2014). Notably, Nox2-
induced mitochondrial ROS formation (for review, see
Dikalov, 2011) may be an important control mechanism in
the coronary microcirculation of patients with CAD. For
example, flow-induced dilation of these vessels requires H2O2
release from the endothelium to activate VSMC hyperpolariz-
ing large conductance Ca21-activated K1 channels (Liu et al.,
2011b), and the accumulation of ROS in coronary ECs exposed
to shear stress is prevented by either Nox2ds-tat or rotenone
(unpublished data). Furthermore, it is now apparent that
MMAIII and DMAIII induce oxidative stress through direct
targeting of mitochondria (Naranmandura et al., 2011) and
the endoplasmic reticulum (Naranmandura et al., 2012),
respectively. Indeed, in cultured human urothelial cells,
MMAIII (although much more potent) takes considerably
longer than AsIII to induce ROS (30 minutes compared with
less than 5 minutes at levels as low as 50 nmol/l and 1 mmol/l,
respectively), prompting these authors to speculate that
MMAIII induces oxidative stress through a mechanism dis-
tinct to that of AsIII (Eblin et al., 2006). How these findings
collectively relate to the chronic development of oxidative
stress in the vasculature in vivo (i.e., under exposure to all
three trivalent arsenicals) is unclear, but observations that
individuals carrying polymorphisms in either p22phox (gain of
function) or Mn-SOD (loss of function) are at increased risk of
arsenic-induced hypertension nevertheless allude to multiple
sources of ROS generation in the context of chronic human
exposure and disease (Hsueh et al., 2005).
Conclusions and Future Directions
This review has described the complex effects of acute and
chronic arsenic exposure on the function of the vascular
endothelium, specifically in relation to regulation of vascular
tone. Compelling evidence suggests that inorganic arsenic
acutely activates and chronically upregulates Nox2, promotes
oxidative stress, reduces NO bioavailability, and suppresses
NOS activity and expression. Future research should focus on
the related effects of arsenic in the microcirculation, where
endothelium-dependent vasodilation is less reliant on NO and
instead mediated primarily by endothelium-derived hyperpo-
larization (EDH) (de Wit and Griffith, 2010; Ellinsworth et al.,
2014a). Indeed, in rabbit iliac arteries H2O2 formed after the
dismutation of AsIII-induced O22 potentiates EDH-type
relaxations and offsets pharmacologically induced NOS in-
hibition (Edwards et al., 2013). However, in this study only
the effects of extremely high AsIII concentrations were
analyzed (Edwards et al., 2013). Nevertheless, the idea that
H2O2 plays a compensatory role is intriguing, because in the
microcirculation of patients with CAD, H2O2 replaces NO as
a major dilator molecule to almost fully preserve endothelial
function (Miura et al., 2001; Phillips et al., 2007; Larsen et al.,
2009; Liu et al., 2011b). However, although vascular function
may be preserved by H2O2 in chronic human disease states,
the fact that the anti-inflammatory and antithrombotic
activity of NO is lost (and that H2O2 is proinflammatory)
has prompted some researchers to question the long-term
benefits of such a substitution in dilator molecule activity (Liu
and Gutterman, 2009; Beyer and Gutterman, 2012; Durand
and Gutterman, 2013).
Furthermore, observations that arsenic potently disrupts
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gap junctional communication through downregulation of all
three dominant vascular connexins (Chou et al., 2007) and in a
tissue-specific manner modulates levels of epoxyeicosatrienoic
acids through alterations in epoxygenase and soluble epox-
ide hydrolase expression (Anwar-Mohamed et al., 2012,
2013, 2014) should also motivate further investigations of
the effects of arsenic on resistance artery function. Indeed,
epoxyeicosatrienoic acids and gap junctions play an integral
role in EDH in many vascular beds (for reviews, see Campbell
and Fleming, 2010; Ellinsworth et al., 2014a,b). Clarification
of these mechanisms will have potentially far-reaching
implications in relation to the understanding of iatrogenic
effects of arsenic during treatment of acute promyelocytic
leukemia, and also the initiation and progression of hyper-
tension and vascular disease due to environmental exposure.
Notably, heavy metal environmental contaminants such as
mercury, cadmium, and chromium are all associated with
endothelial dysfunction and cardiovascular risk through
mechanisms involving oxidative stress and Nox2 (Wiggers
et al., 2008; Almenara et al., 2013; Kukongviriyapan et al.,
2014).
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Ellinsworth.
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Address correspondence to: David C. Ellinsworth, Bristol Heart Institute,
University of Bristol, Queens Building Level 7, Bristol Royal Infirmary, Upper
Maudlin Street, Bristol, BS2 8HW, UK. E-mail: davidellinsworth@gmail.com
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