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Cite this: DOI: 10.1039/c7mt00201g
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Selenium-mediated arsenic excretion in mammals:
a synchrotron-based study of whole-body
distribution and tissue-specific chemistry†
Olena Ponomarenko, ‡a Paul F. La Porte,‡bcd Satya P. Singh,a George Langan,e
David E. B. Fleming, f Julian E. Spallholz,g Mohammad Alauddin,h Habibul Ahsan,i
Selim Ahmed,j Jürgen Gailer, k Graham N. George *al and
Ingrid J. Pickering *al
Arsenicosis, a syndrome caused by ingestion of arsenic contaminated drinking water, currently affects
millions of people in South-East Asia and elsewhere. Previous animal studies revealed that the toxicity of
arsenite essentially can be abolished if selenium is co-administered as selenite. Although subsequent
studies have provided some insight into the biomolecular basis of this striking antagonism, many details of
the biochemical pathways that ultimately result in the detoxification and excretion of arsenic using
selenium supplements have yet to be thoroughly studied. To this end and in conjunction with the recent
Phase III clinical trial ‘‘Selenium in the Treatment of Arsenic Toxicity and Cancers’’, we have applied
synchrotron X-ray techniques to elucidate the mechanisms of this arsenic–selenium antagonism at the
tissue and organ levels using an animal model. X-ray fluorescence imaging (XFI) of cryo-dried whole-body
sections of laboratory hamsters that had been injected with arsenite, selenite, or both chemical species,
provided insight into the distribution of both metalloids 30 minutes after treatment. Co-treated animals
Received 8th July 2017,
Accepted 9th October 2017
DOI: 10.1039/c7mt00201g
showed strong co-localization of arsenic and selenium in the liver, gall bladder and small intestine. X-ray
absorption spectroscopy (XAS) of freshly frozen organs of co-treated animals revealed the presence in liver
tissues of the seleno bis-(S-glutathionyl) arsinium ion, which was rapidly excreted via bile into the intestinal
tract. These results firmly support the previously postulated hepatobiliary excretion of the seleno bis-(S-
glutathionyl) arsinium ion by providing the first data pertaining to organs of whole animals.
Significance to metallomics
Chronic arsenic poisoning through consumption of arsenic-laden groundwater, or arsenicosis, is a problem for tens of millions of people in Bangladesh and
surrounding areas. We have previously suggested that dietary selenium supplements might be used as a palliative for arsenicosis. By examining the microscopic
distribution and molecular association of arsenic and selenium, this paper provides direct evidence, at an organ and tissue level, that selenium binds to arsenic
in vivo in mammals, facilitating its excretion from the body. Our results provide a foundation for clinical use of selenium supplements to treat arsenicosis.
Molecular and Environmental Science Research Group, Department of Geological
Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada.
Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA
Emory University School of Medicine, Emory University, Atlanta, GA 30322, USA
Division of Hematology-Oncology, University of California Los Angeles, CA 90095, USA
Department of Surgery, University of Chicago, Chicago, IL 60637, USA
Department of Physics, Mount Allison University, Sackville, NB, E4L 1E6, Canada
Nutritional Sciences, Texas Tech University, Lubbock, TX 79409, USA
Chemistry Department, Wagner College, Staten Island, NY 10301, USA
Department of Health Sciences, University of Chicago, Chicago, IL 60637, USA
School of Medicine, University Malaysia Sabah, Sabah 88400, Malaysia
Department of Chemistry, University of Calgary, Calgary, AB, T2N 1N4, Canada
Department of Chemistry, University of Saskatchewan, Saskatoon, SK, S7N 5C9, Canada
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mt00201g
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2017
Worldwide, close to 150 million individuals consume drinking
water from aquifers that are geologically contaminated with
inorganic arsenic at concentrations exceeding the upper limit
of 0.010 mg L1 as recommended in the WHO guidelines.1–3
This number includes approximately 57 million people in
Bangladesh alone. Long-term consumption of arsenic in drinking
water results in arsenicosis,4 a syndrome featuring hypermelanosis
and hyperkeratosis, vascular and endocrine pathologies, several
types of cancer and increased mortality.5–8
There are no proven preventive procedures or treatments for
arsenicosis-related cancers. However, it is known from several
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Fig. 1 XFI elemental distributions in whole-body sections of hamster prepared from the control (sham), and after dosing with selenite (Se), arsenite (As),
or arsenite + selenite (Se + As) in the ratio As : Se = 5 : 1. (A) Gross view of lyophilized sections; elemental distributions of (B) Se and (C) As; and (D)
elemental distributions of Ca (green), Fe (red) and Zn (blue) displayed as tricolor images. LV, liver; SI, small intestine; HT, heart; LG, lungs; D, diaphragm.
Arsenic and selenium correlation coefficients for the Se + As specimen are presented in Table 2, with elemental tissue concentrations in Table S3 (ESI†).
animal studies that selenium can counteract arsenic toxicity.9,10
Arsenic is not thought to be essential for human health at any
level.4 Conversely, selenium is an essential ultratrace element
which needs to be ingested in the human diet in sufficient
amounts to satisfy the endogenous biosynthesis of essential
selenoproteins, such as a glutathione peroxidase, thioredoxin
reductase and thyroxine deiodinase.11,12
The striking trace element antagonism between inorganic
arsenic and selenium compounds was first reported by Moxon
in 1938.13 Since then it has been the subject of over 35 animal
studies,10,13–18 demonstrating repeatedly that an otherwise
lethal dose of selenite effectively counteracted the toxic effects
of an otherwise lethal dose of arsenite. In 1966, Levander and
Baumann reported that in rats arsenite increased the biliary
elimination of selenium following administration of selenite
and postulated a ‘‘reaction between arsenic and selenium
forming some sort of detoxication conjugate readily excreted
into the bile’’. However, at that time evidence supporting the
mechanism of this arsenic–selenium antagonism could not be
obtained.15,19 The advent of synchrotron-based techniques
enabled the study of the underlying biomolecular basis of this
antagonism and revealed the in vivo formation of the seleno bis(S-glutathionyl) arsinium anion, [(GS)2AsSe], in blood followed by
its rapid excretion in bile via the hepatobiliary system.16
In our prior work with rabbits and rats we have applied X-ray
absorption spectroscopy (XAS) to demonstrate the in vivo formation
of [(GS)2AsSe], in which arsenic is covalently bound to
selenium.16,20–23 This species is formed in hepatocytes and
possibly erythrocytes, and is rapidly excreted from the liver into
bile,16,17,23 most likely through the activity of the ATP-binding
cassette (ABC) transporter family member multidrug resistance
protein 2 (MRP2 or ABCC2).24 It is likely that the formation
of [(GS)2AsSe] in the bloodstream and its rapid excretion:
(1) reduces the availability of circulating arsenicals for tissue
deposition;25 (2) precludes the methylation of AsIII in the liver26
to the more carcinogenic methylated species monomethylarsonous
acid (MMAIII) and dimethylarsinous acid (DMAIII);27 and (3)
mobilizes deposited arsenic from peripheral tissues.17
Informed by these discoveries, clinicians have examined
whether the provision of dietary selenium in the form of selenite
might be able to detoxify arsenite in humans before the latter
can invade tissues to cause organ-based toxicities. Inadequate
dietary intake of selenium and accelerated selenium depletion
by arsenic are possible contributing factors to arsenicosis.16,28
Two Phase II trials, in China and Bangladesh, have suggested
some clinical benefits from selenium supplementation in
arsenicosis patients.29,30 However, many details of the physiological and biochemical pathways leading to inactivation and
excretion of arsenic using selenium supplements have yet to be
thoroughly studied.
In conjunction with the recent Phase III clinical trial ‘‘Selenium
in the Treatment of Arsenic Toxicity and Cancers’’ (SETAC),31 we
devised a strategy using synchrotron techniques to elucidate
mechanisms of arsenic–selenium counteraction at the tissue and
organ levels using an animal model. We employed wide format
X-ray fluorescence imaging (XFI) of flash frozen and lyophilized
whole-body sections of laboratory hamsters to study the distributions
of arsenic and selenium in different organs after intravenous
injections of equimolar concentrations of arsenite and selenite.
In combination with this, we used X-ray absorption spectroscopy to determine chemical speciation of arsenic and selenium,
measured on freshly frozen tissues excised from adjacent sections
of injected animals in order to detect otherwise unstable selenium
and arsenic metabolites.
X-ray fluorescence imaging
Representative XFI elemental maps of scanned specimens from
each of the four treatment groups are shown in Fig. 1. The
elemental concentrations of Ca, Mn, Fe, Cu, Zn, As and Se
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(mg per g of dry weight, mg g1) for the specimens’ liver, lungs,
heart, throat, stomach and intestines are presented in Table S3
Control. The control hamster (Fig. 1 and 2a) gave endogenous
trace selenium concentrations that were highest in the liver
(mean = 7 mg g1 dry weight), followed by the heart and lungs
(each B2 mg g1). Arsenic concentrations were very small, the
highest being in the intestines (B0.1 mg g1). In the control
specimen, the distributions of arsenic and selenium throughout
the body tissues were not spatially correlated (r 2 o0.01). The
concentrations of other trace elements (Table S3, ESI†), such as
Zn and Fe, were close to literature values32,33 for hamsters.
Selenite dosing. Selenite-only dosing (Fig. 1 and 2b) resulted
in selenium being distributed throughout the hamster body,
accumulating to particularly high levels in the liver (157–240 mg g1),
heart (149 mg g1), and lungs (56 mg g1), with around 43% of
total selenium in the section accumulated in these tissues.
Arsenic was close to or below detection limits with no apparent
correlation in spatial distributions of trace arsenic and injected
selenium (for all organs, r 2 o 0.01).
Arsenite dosing. Arsenite-only dosing (Fig. 1 and 2c) led to
arsenic being distributed throughout the vascularized soft
tissues of the hamster, with the highest concentrations in the
gallbladder (250 mg g1), liver (150–200 mg g1), small intestine
(60–88 mg g1) and lungs (90 mg g1). These organs accumulated
approximately 50% of the total arsenic in the section. Similar to
the control, trace levels of endogenous selenium were observed
in many tissues. The highest levels were apparent in the liver
(6 mg g1), in particular, in the tissues surrounding the bile
ducts (B7 mg g1). Similar to the control and the selenite-dosed
animals, the spatial correlation between arsenic and selenium
was very low, with the highest values of r 2 found in the liver
(r 2 B 0.13) and small intestine (r 2 B 0.1).
Dual dosing. For the hamster dual dosed with both arsenic
and selenium at a 1 : 1 molar ratio (Fig. 3a), co-localization of
arsenic with selenium was visually apparent throughout the
whole body, with the preponderance of whole section arsenic (66%)
and selenium (60%) localized in the liver (As = 91–228 mg g1,
Se = 85–312 mg g1), the gallbladder/bile duct area (As = 355 mg g1,
Se = 414 mg g1) and the proximal small intestine (As = 243 mg g1,
Se = 350 mg g1). Correlation coefficients (r 2) between arsenic and
selenium, together with their average molar concentration ratios,
are presented in Table 1 for various tissues. There was a high degree
of co-localization of arsenic and selenium in the gallbladder and the
bile ducts (r 2 = 0.90), liver (r 2 = 0.64–0.73) and the small
intestine (r 2 = 0.72–0.86). Since arsenic and selenium were
injected intravenously rather than orally gavaged, their presence
in the small intestine must be attributed to their biliary excretion.
The As : Se molar concentration ratio (Table 1) was closer to 1 : 1
in these organs, compared with tissues of the lung (As : Se = 0.69),
muscle (As : Se = 0.58), throat (As : Se = 0.77), and brain
(As : Se = 0.20).
The non-equimolar dual dosing (As : Se = 5 : 1, Fig. 1 and 3b)
shows sharper tissue-specific differences in the distributions of
these elements (Table 2) compared with the 1 : 1 dosing. Liver,
small intestines and bile duct together accumulated 40 and
This journal is © The Royal Society of Chemistry 2017
34% of the total observed As and Se, respectively, while the lung
tissues accumulated an additional 15% of total As and 25% of
total Se. Similar results were obtained in our related study
using portable XRF measurements of As and Se in a hamster
injected with a molar As : Se ratio of B2 : 1 (after correction of
the concentrations reported in that paper in relation to wet
tissue weight).34
Patterns of arsenic accumulation in different organs for
arsenite-only and dual-dosing (As + Se) indicate that the hepatobiliary pathway of arsenic excretion was predominant when
selenium was co-administered. These data clearly demonstrate
that simultaneous administration of high concentrations of
selenite and arsenite in blood opens the pathway of hepatobiliary
excretion of arsenic from the body in form of the [(GS)2AsSe]
X-ray absorption spectroscopy
We used bulk near-edge XAS to determine the arsenic and
selenium speciation in rapidly frozen organ samples dissected
from spare sections of the same animals shown in Fig. 1–3.
This method was chosen since [(GS)2AsSe] is expected to be
unstable under conditions of lyophilization. Flash freezing in
liquid nitrogen followed by XAS measurements at liquid helium
temperatures has been shown to arrest degradation of the
unstable redox-active thiol compounds in brain tissues.35 Previous
rapid freezing of blood or bile samples allowed us to determine the
key component in the physiology of the arsenic–selenium
antagonism, [(GS)2AsSe].16,17,23 Near-edge XAS is sensitive
to the chemical environment (oxidation state and electronic
structure, neighbouring atom geometry) of the absorbing
atom.36 Se K and As K near-edge XAS of liver and gallbladder
(bile) tissues of animals following different treatments were
analyzed by fitting to a linear combination of standard spectra
(Fig. 4, Tables 3, 4 and Fig. S1, ESI†).
Control. The Se K near-edge spectrum of the liver of the
control animal (i.e. dosed with sham) is shown in Fig. S3 (ESI†),
together spectra of selected selenium standards. Since only
trace levels of selenium are found in the liver of control animals
(Table S1, ESI†), the spectrum has low signal to noise. However,
qualitatively the XAS shows the most similarity with that of
standard aqueous solutions (pH = 7.5) of selenocysteinate and
selenomethionine, and may arise from a mixture of compounds
containing Se–C bonds which are naturally abundant in selenoproteins of liver tissues.37
Selenite dosing. The Se-only dosed animal yielded a Se K
near-edge XAS of liver that could be best fit by a combination of
selenium-diglutathione, GS–Se–SG (72%) and seleno-cysteinate,
[Cys-Se] (28%) species (Table 4 and Fig. 4f). Since XAS is
sensitive to the type of bonding environment of the absorbing
atom, these results can be interpreted as a prevalence of RS–Se–SR
environment for selenium represented by GS–Se–SG standard,
and a selenide [RSe] or a selenopersulfide type of environment
[RS–Se] represented in fitting by deprotonated seleno-cysteinate
[Cys–Se]. While excretion of methylated selenium compounds via
lungs and kidneys as well as selenosugars via kidneys are regarded
as the main selenite detoxification pathways,38 especially during
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Fig. 2 High resolution XFI of arsenic (red), selenium (blue), and calcium (green) in sagittal cross-sections of hamster specimens: (a) control; (b) dosed
with selenite only; (c) dosed with arsenite only. The As and Se intensities are on a common scale for all of these panels, with higher concentrations of As
and Se denoted by brighter colors. Elemental tissue concentrations are presented in Table S3 (ESI†).
exposure to prolonged sub-acute or dietary concentrations, our
experiments suggest that 30 minutes of acute exposure to intravenously injected selenite is insufficient for products of selenium
methylation in tissues to accumulate to sufficient proportions of
the total selenium to be detected by XAS.
Arsenite dosing. For the As-only dosed animal, the As K XAS
of liver could be best fit to arsenic-triglutathione As(GS)3 (61%),
MMAIII (25%) and arsenate [AsO4]3 (14%) (Table 3 and Fig. 4c). The
major component, As(GS)3, is a key intermediate in mammalian
metabolism of arsenite found in bile of mammals.39 Together with
MMAIII, As(GS)3, methylarsonous diglutathione, and dimethylarsinous glutathione possibly represent the main arsenic species
that are transported from the liver to the kidneys for urinary
excretion.40 The minor components, MMAIII and [AsO4]3 (Table 3
and Fig. 4c), could be interpreted as a mixture of products of the
sequential oxidation and methylation reactions involved in the
methylation pathway, resulting in formation of methylated AsIII
and AsV metabolites.41 Similar to the control animal, Se K XAS of
liver from the As-only dosed animal (Fig. S1, ESI†) has low
signal to noise but qualitatively appears most consistent with
selenoproteins of the endogenous selenium pool naturally
abundant in liver. Seemingly, the time elapsed since the bolus
exposure to arsenite (30 min) is not sufficient for the release of
significant levels of endogenous selenium from selenoproteins
to form [(GS)2AsSe].
Dual dosing. After equimolar dosing with As and Se, essentially
all arsenic and selenium in the liver was present as [(GS)2AsSe]
(Tables 3, 4 and Fig. 4b, e). This result is consistent with
the hepatobiliary pathway of selenium-mediated excretion of
arsenic.16,17,21,23 In the gallbladder (bile) after dual dosing with
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Fig. 3 High resolution XFI of arsenic (red), selenium (blue), and calcium (green) in sagittal cross-sections of hamster specimens with dual (arsenite +
selenite) dosing: (a) dosed with As : Se = 1 : 1 (sample #1335); (b) dosed with As : Se = 5 : 1 (sample #1337). Magenta indicates co-localization of Se and As.
Higher concentrations of As and Se are denoted by brighter colors. Tissue arsenic and selenium concentrations, ratios and correlation coefficients are
given in Tables 1 and 2.
Table 1 Mean tissue concentrations of As and Se (mg g1) and their ratios
by mass (mg mg1) and by moles (mol mol1) in hamster specimen #1335
dual dosed with As : Se 1 : 1. Squared coefficient of linear correlation
between Se and As distributions, r 2, is shown in the last column
Table 2 Mean tissue concentrations of As and Se (mg g1) and their ratios
by mass (mg mg1) and by moles (mol mol1) in hamster specimen #1337
dual dosed with As : Se 5 : 1. Squared coefficient of linear correlation
between Se and As distributions, r 2, is shown in the last column
[mg g1]
[mg g1]
[mg mg1]
[mol mol1]
[mg g1]
[mg g1]
[mg mg1]
[mol mol1]
Whole slice
Liver 1
Liver 2
Bile duct
Intestine 1a
Intestine 1b
Intestine 2
Whole slice
Liver 1
Liver 2
Bile duct
Intestine 1a
Intestine 1b
Intestine 2
As : Se = 5 : 1, consistent with the excess dose of arsenic, some
28% of total arsenic was bound to the available selenium with
the balance of arsenic present as As(GS)3 species (Tables 3, 4
and Fig. 4a, d). Conversely, essentially all of the selenium was
bound to arsenic in form of [(GS)2AsSe].
Discussion and conclusions
Implications for arsenic–selenium antagonism
Our previous work on rabbits and rats established the in vivo
formation and rapid biliary excretion of [(GS)2AsSe].16,17,23
This journal is © The Royal Society of Chemistry 2017
We hypothesized that this pathway provides the biomolecular
basis for the arsenic–selenium antagonism noted in many
animal studies since the 1930s. Our previous experiments with
rats confirmed that [(GS)2AsSe] rapidly appears in the bile of
arsenite-intoxicated animals treated with selenite.23 Unlike
rabbits, hamsters or humans, rats do not possess gallbladders
where compounds pool and may decompose. In the present
work we used a hamster animal model which enables additional
conclusions to be drawn.
This study used a combination of two different synchrotron
techniques. Wide format synchrotron X-ray fluorescence imaging
(XFI) allowed direct visualization and determination of arsenic
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Fig. 4 As K near-edge XAS (left panels) from: (a) gallbladder, specimen #1337 (dual dosing As : Se 5 : 1); (b) liver, specimen #1335 (dual dosing As : Se 1 : 1);
(c) liver, specimen #1338 (dosed with arsenite only). Se K near-edge XAS (right panels) from: (d) gallbladder, specimen #1337; (e) liver, specimen #1335;
(f) liver, specimen #1341 (dosed with selenite). Each panel shows the data (open circles), the linear combination fit (green) and the components of the fit,
scaled according to their percentage contribution to the fit as identified in the legend. Numerical results are summarized in Tables 3 and 4.
Table 3 Linear combination analysis of As K near-edge XAS in gallbladder
and liver tissues for different dosing scenarios, showing types of arsenic
chemical environment [%]. Percentage values were determined from linear
combination of standard components. e values are 3 the estimated
standard deviations obtained from the diagonal elements of the covariance matrix. NS, not significant
Table 4 Linear combination analysis of Se K near-edge XAS in gallbladder
and liver tissues for different dosing scenarios, showing types of selenium
chemical environment [%]. Percentage values were determined from linear
combination of standard components. e values are 3 the estimated
standard deviations obtained from the diagonal elements of the covariance
matrix. NS, not significant
Arsenic standard components [% e]
Gallbladder /As + Se
Liverb/As + Se
28 2
100 3
72 2
61 3
25 4
Selenium standard components [% e]
14 2
Gallbladder /As + Se
Liverb/As + Se
100 2
92 2
72 3
28 3
Specimen #1337 (dual dosing As : Se = 5 : 1). b Specimen #1335 (dual
dosing As : Se = 1 : 1). c Specimen #1338 (arsenite-only dosing).
Specimen #1337 (dual dosing As : Se = 5 : 1). b specimen #1335 (dual
dosing As : Se = 1 : 1). c specimen #1341 (selenite-only dosing).
and selenium elemental distributions in flash-frozen and then
lyophilized full-body hamster sections. This was used in
conjunction with synchrotron X-ray absorption spectroscopy
(XAS) in hydrated, flash-frozen tissues of interest (in this case,
liver and gallbladder excised from leftover sections of the
corresponding animals), conducted under cryogenic conditions
for chemical speciation studies of otherwise unstable selenium
and arsenic metabolites. Thus, to our knowledge, this is the first
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study in the field of mammalian metabolism of heavier elements
based on the combination of these two techniques. Our approach
can be applied to detailed investigations of metal interactions at a
broad range of hierarchical levels, from whole-body pharmacodynamics to tissue/cell chemistry, using either acute or chronic
exposure scenarios.
Equimolar dosing with As and Se leads to significant 1 : 1
co-localization of arsenic and selenium in the liver, gallbladder,
and small intestine, consistent with the hepatobiliary excretion
pattern postulated by prior experiments on rats and
rabbits.9,10,16,20–23,41 Upon injection, As and Se interact in the
liver, and possibly in the bloodstream, forming As–Se species
which accumulate in the liver and gallbladder of hamsters and
also are found in high concentrations in the intestinal tracts of
injected animals. XAS of liver tissues and gallbladder following
equimolar dosing demonstrated formation of [(GS)2AsSe] as
the main As species. Our results show that the liver excretes
nearly all arsenic in the form of [(GS)2AsSe], suggesting that
any decomposition may not be expected to occur until exposure
to oxygen in the gastrointestinal lumen.42
The human hepatic efflux pump MRP2 was recently identified
in vitro24 as a high-affinity transporter of [(GS)2AsSe], which
supports our prior observations of biliary excretion of [(GS)2AsSe]
in rats and rabbits. While our work in rabbits and rats had already
suggested that arsenic and selenium are preferentially co-localized
to the liver and excreted through the hepatobiliary tree, the results
herein of XFI and tissue-level XAS analysis in hamsters provide
graphic illustration of this hepatobiliary excretion pattern. The
converging evidence from these experiments suggests that
[(GS)2AsSe] is formed within minutes in the blood and liver
of hamsters and is rapidly excreted via bile into the small
intestines. The predominance of this effect in the previous
experiments in a rabbit model indicates that it represents the
primary pathway for selenium’s detoxification of arsenic in
We demonstrated that the major arsenic compound found
in liver tissues of hamsters treated with arsenite alone is trisS-glutathionyl arsenic(III) [As(GS)3]. In experiments using
sandwich-cultured hepatocytes, the arsenic–glutathione conjugates
As(GS)3, CH3As(GS)2 as well as [(GS)2AsSe] were shown to be the
substrates of the multidrug resistance protein 2 (MRP2).24 These
authors suggested that the remarkable protective properties of
selenium against arsenic toxicity are based on two factors: a higher
stability of [(GS)2AsSe] at the pH of bile compared to As(GS)3 and
CH3As(GS)2; and a higher affinity of [(GS)2AsSe] for MRP2
compared with As(GS)3, possibly leading to the preferential
excretion of the former species in bile. These data suggest that
the formation of [(GS)2AsSe] prevents As from undergoing
enterohepatic cycling and directs it via the hepatobiliary pathway to more efficient detoxification.24
In conclusion, our findings provide a compelling explanation
for 80 years of observations that selenium counters arsenic
toxicity in a variety of animal models. Furthermore, this work
provides a physiological justification for the clinical use of
selenium supplements to treat arsenicosis. Forthcoming clinical
trials should address the effectiveness of different chemical
This journal is © The Royal Society of Chemistry 2017
forms of selenium in countering arsenicosis as well as the
pharmacokinetic profile and optimal dosing for [(GS)2AsSe]
Materials and methods
Choice of arsenite and selenite
Our choice of arsenite and selenite was based upon several
considerations focused on the overall goal of finding efficient
palliatives against arsenicosis. (1) Inorganic arsenicals in drinking
water are proven human Group 1 carcinogens.43 Arsenite (AsIII) is a
prevalent form in As-contaminated drinking water sources, found
in shallow water wells with reducing conditions (low oxygen and
high organic content) in Bangladesh, one of the countries most
affected by arsenicosis worldwide.44–47 (2) Arsenite is transported
through the gastrointestinal barrier more efficiently compared to
other arsenic species.48 (3) Inorganic arsenate (AsV) is also of
concern, being found mostly in open water sources with oxidizing
conditions (rivers, lakes).49 However, uptake of arsenate into
the cell is less efficient compared to that of arsenite. Moreover,
arsenate is reduced to AsIII species under physiological
conditions.50,51 (4) Arsenite and selenite directly react with
endogenous glutathione to form the seleno-bis(S-glutathionyl)
arsinium ion16 which is the molecular basis for the antagonism
of interest. Had we chosen arsenate and selenate it would be
much more difficult to determine when the As–Se compound
would be formed, because arsenate and selenate would have to
be reduced to arsenite and selenite first by corresponding
arsenate and selenate reductases in the liver, which takes time,
as indicated by our previous experiments.52
Choice of animal species
The responses among mammalian species to toxic and carcinogenic substances including arsenic compounds differ greatly,53–55
with the differences attributed to reactivity of hemoglobin sulfhydryl residues.53 Previous studies suggested the binding affinities of
rat hemoglobin sulfhydryl groups with trivalent arsenic species
results in both greater retention of arsenicals within red blood
cells, with correspondingly lower arsenical distribution to tissues,
compared to that in humans.56 Hence, rats and mice were not
considered due to the high proportion of cysteine residues in their
hemoglobin,53,56–58 distorting the pharmacokinetic profile for
arsenic when compared with humans and other mammals.
Furthermore, rats lack gallbladders and therefore would not show
the anticipated concentration of [(GS)2AsSe] in this organ. In
contrast to rats, hamsters do possess a gall bladder and therefore
represent an animal model that more closely resembles the
metabolism in humans. Larger mammals, such as rabbits, were
not considered due to the difficulty of imaging much larger
sections in the XFI apparatus. Hamsters were therefore selected
due to (1) their size, (2) the presence of a gallbladder, and (3) the
more human-like composition of their hemoglobin compared to
that of rats57 and mice.58
The statistical outcomes of chronic exposure of human populations to arsenic are influenced by gender.59–65 While not being
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completely understood, these effects are related to long-term
exposure. In contrast, the design of our experiments is based upon
previous observations of the fast, non-enzymatic formation of the
seleno-bis(S-glutathionyl) arsinium ion16 in blood,20 and in abiotic
in vitro solutions in presence of glutathione.22 This study was
designed to better understand the effect of selenite on the
metabolism of arsenite at the systemic level. Our literature review
suggested that there would be no significant difference in arsenite
or selenite pharmacokinetics for opposite genders. We therefore
concluded that for the short exposure time (30 min) and concentration scale (mM) of our experiments, the hormonal variations
within one sex should not affect the systemic toxicity, and studied
it using single gender in a standard (normal) type Lakeview/LVG
Syrian hamster. Furthermore, due to constraints in sample preparation and synchrotron resolution, it was preferable to use larger
specimens. Since female hamsters of a given age are roughly 25%
larger than their male counterparts, we used females for these
Specimen preparation
Female Lakeview Golden/LVG Syrian hamsters weighing approximately 150 g were purchased from Charles River Laboratories with
indwelling jugular catheters. After 72 hours of acclimation, hamsters
were anesthetized with isoflurane gas, and dosed intravenously
(bolus IV) as follows: (a) sham and sham in the form of normal
saline; (b) sham and arsenic, dosed at 2.40 mg kg1 from a solution
of sodium arsenite; (c) sham and selenium, dosed at 2.52 mg kg1
from a solution of sodium selenite; and (d) arsenic and selenium,
dosed in the equimolar ratio As : Se = 1 : 1 (2.40 mg kg1 of
arsenic and 2.52 mg kg1 selenium), and in the ratio As : Se = 5 : 1
(2.40 mg kg1 of arsenic and B0.5 mg kg1 of selenium).
After 30 minutes, hamsters were euthanized with a pentobarbital,
and immediately were flash-frozen in liquid nitrogen. Subsequently,
animals were encased in Tissue-Tek OCT embedding compound
(Sakura Finetek, California), and sectioned while frozen with a
DeWalt DW716R 1200 miter saw (DeWalt Industrial Tool Co.,
Maryland). Sections from the central part of the body (along the
vertebrae) were prepared for XFI by lyophilization for over
12 hours. The average thickness of each lyophilized section,
measured using calipers at 6 locations per section (see ESI†) was
in the range 1 to 2 mm. Remaining sections were preserved at
80 1C, from which frozen tissues from different organs were
subsequently excised for bulk XAS analysis. Dissection of frozen
organs was performed very quickly in a cold room environment
to preserve unstable metabolites. Frozen tissues were tightly
packed into 2 mm-pathlength Lucite cells, with metal-free Mylar
tape for windows, and were stored at liquid nitrogen temperatures
until XAS experiments were performed. All animal work was
conducted at the University of Chicago. The experiment was
approved by the University of Chicago Institutional Animal Care
and Use Committee as protocol no. 72167.
X-ray fluorescence imaging
XFI measurements66 were conducted at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 10-2, optimized
for wide format, continuous-scanning, large-aperture (100 mm)
X-ray fluorescence imaging (XFI) of large specimens. The scan
time is reduced to approximately 30 s cm2, permitting multielement XFI scans sampled at a rate of few milliseconds per
pixel. This technique has been used to study heterogeneous
and fragile large samples, from deciphering hidden pages of
the of the Archimedes Palimpsest,67 and analyzing the feather
and bone chemistry of the Archaeopteryx,68 to mapping trace
elements in autopsied human brains.69
The XFI experimental setup is shown in Fig. S2 (ESI†). The
intensity of the incident beam (13 540 keV) was monitored with
a nitrogen gas-filled ion chamber. Each lyophilized animal slice
was placed on a metal-free plastic sheet, framed by metalfree double-sided adhesive tape, and sealed with a window of
polypropylene film. Samples were mounted vertically on a
motorized stage at 451 to the incident X-ray beam and rasterscanned in the beam, using a 50 ms per pixel dwell time, with
stepsize of 250 or 125 mm for the initial or higher resolution
imaging. A germanium detector was positioned at a 901 angle to
the beam in the horizontal plane. Data were collected with the
MICROSCAN program with the XMAP GUI visual interface
software. Data reduction, performed using custom Fortran-95
code, included subtracting: (1) spectrally overlapping signals
from matrix elements with close atomic numbers (e.g. arsenic
in the case of selenium); (2) background signal from trace
elements in the mount, determined from mount-only measurements; and (3) the ‘‘tail’’ of the Compton scatter signal, determined
from blank standard measurements. Concentrations of elements of
interest (Ca, Mn, Fe, Cu, Zn, As, Se) were calibrated using certified
highly uniform thin film standards on 6.3 mm-thick mylar substrates
(Micromatter, Vancouver, BC, Canada). The pixel-by-pixel concentrations of elements (mg g1 dry weight) accounting for sample
thickness effects were calculated using an algorithm70 described in
ESI† and coded in Matlab. Images were graphically rendered for
presentation using the MicroAnalysis ToolKit program.71
X-ray absorption spectroscopy
XAS data were collected at SSRL beamline 7–3 (Fig. S3, ESI†),
using Si(220) monochromator crystals. Harmonic rejection was
accomplished by setting the energy cutoff of the upstream
Rh-coated bent flat mirror to 15 keV. Incident and transmitted
X-ray intensities were monitored using nitrogen gas-filled ionization
Near-edge XAS data were measured as the primary fluorescence excitation spectrum using a 30-element germanium array
detector (Canberra Industries, Meriden, CT). The samples were
maintained at approximately 10 K in a liquid helium flow
cryostat CF1204 (Oxford Instruments, Concord, MA). The arsenic
spectra were energy-calibrated with reference to an elemental
grey arsenic foil measured simultaneously with the data, the
lowest energy inflection of which was assumed to be 11867.0 eV.
The selenium spectra were calibrated similarly using a hexagonal
elemental selenium foil (12658.0 eV). Data reduction and analysis
used the EXAFSPAK suite of computer programs.72
Spectra were normalized relative to the As K or Se K edgejumps using the EXAFSPAK program BACKSUB. The PCA and
TARGET subroutines were used to pre-select the reference
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standard spectra for the subsequent least-squares fitting. The
decomposition of the As K and Se K near-edge spectra from
frozen hamster tissues to linear combinations of standard spectra
was performed using DATFIT. The criterion for a standard
component to be included in the fit is that the fitted fraction
must be greater than 3 times the estimated standard deviation
(corresponding to the 99% confidence limit) obtained from the
diagonal elements of the covariance matrix. Reference arsenicand selenium-containing compounds tested for fitting are
listed in Table S4 (ESI†).
Conflicts of interest
There are no conflicts of interest to declare.
We thank T. C. MacDonald, M. Gallego-Gallegos, M. J. Hackett,
N. V. Dolgova, K. H. Nienaber, A. K. James and S. Nehzati for
assistance in XFI and XAS experiments. Work at the University
of Saskatchewan was funded by awards from the Canadian
Institutes of Health Research (CIHR) (FRN68849 to G. N. G. and
I. J. P.), the Natural Sciences and Engineering Research Council
of Canada (NSERC) (RGPIN 04632-2014 to G. N. G. and RGPIN
05810-2016 to I. J. P.), by the Government of Saskatchewan
Innovation and Science Fund (to I. J. P.) and by the Canada
Foundation for Innovation (to I. J. P.). G. N. G. and I. J. P. are
Canada Research Chairs. O. P. is a Research Associate in the
CIHR Training grant in Health Research Using Synchrotron
Techniques (CIHR-THRUST). J. E. S. and P. F. L. P. acknowledge
grant support from the National Cancer Institute/National
Institutes of Health (NIH) (grant 1R21CA117111-01) and American
Cancer Society (grant ROG-06-098-01). Work at the University of
Chicago was funded by the NIH (R01CA107432 and R01CA102484
to HA). Work at Mount Allison University was funded by NSERC
(RGPIN-261523 to D. E. B. F.). Use of the Stanford Synchrotron
Radiation Lightsource, SLAC National Accelerator Laboratory, is
supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences under Contract No. DE-AC0276SF00515. The SSRL Structural Molecular Biology Program is
supported by the DOE Office of Biological and Environmental
Research, and by the NIH, National Institute of General Medical
Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not
necessarily represent the official views of NIGMS or NIH.
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