# Methylated Trivalent Arsenic-Glutathione Complexes  are More Stable than their Arsenite Analog

Authors: Andrew J. Percy; Jürgen Gailer
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

The trivalent arsenic glutathione complexes arsenic triglutathione,
methylarsonous diglutathione, and dimethylarsinous glutathione are key intermediates
in the mammalian metabolism of arsenite and possibly represent the arsenic species
that are transported from the liver to the kidney for urinary excretion. Despite this, the
comparative stability of the arsenic-sulfur bonds in these complexes has not been
investigated under physiological conditions resembling hepatocyte cytosol. Using
size-exclusion chromatography and a glutathione-containing  phosphate buffered saline
mobile phase (5 or 10 mM glutathione, pH 7.4) in conjunction with an
arsenic-specific detector, we chromatographed arsenite, monomethylarsonous acid, and
dimethylarsinous acid. The on-column formation of the corresponding arsenic-glutathione
complexes between 4 and37∘C revealed that methylated arsenic-glutathione complexes are more
stable than arsenic triglutathione. The relevance of these results with regard to the metabolic
fate of arsenite in mammals is discussed.


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## Body

## 1. Introduction

The metalloid arsenic (As) ranks 53rd amongst elements in the Earth’s crust [1], where it is predominantly found in sulfidic ores, such as arsenopyrite
(FeAsS) and orpiment (As2S3) [2]. Natural and anthropogenic activities, however, mobilize geogenic arsenic
into the aquatic environment, including drinking water, where arsenite (As(III)) and/or arsenate (As(V))
are the most prevalent oxyanions [2]. Because the chronic ingestion of only 50–200 μg/day of inorganic arsenic is associated with cancers of the skin, the liver, the lungs,
the kidneys, and the bladder in humans  [3–7], the exposure of humans to
concentrations of inorganic As in drinking water that are unsafe for human
consumption currently affects∼100 million people [8]. In fact, the low-level As
poisoning tragedy that is currently unfolding in parts of India and Bangladesh
has been referred to as the largest mass poisoning in history [9, 10].Investigations carried out in the 1970s,
whichaimed to identify As-containing metabolites in human urine, indicated two pentavalent organoarsenicals monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)), in addition to As(III) and As(V) [11]. More recently, two additional trivalent organoarsenicals—monomethylar-sonousacid [MMA(III), CH3As(OH)2,
Figure 1(b), [12–16]] and dimethylarsinous acid [DMA(III),
(CH3)2AsOH, Figure
1(c), [12, 14, 15]]—have also been identified (we note that other As-containing metabolites, such as thiodimethylarsenopropanoic acid and thiodimethylarsenobutanoic acid, have been recently identified in human urine as metabolites of ingested arsenolipids [17]).Solution species at pH 7.4 of (a) arsenite, As(III), (b) monomethylarsonous
acid, MMA(III), (c) dimethylarsinous acid, DMA(III), (d) arsenobetaine, AsB, (e) glutathione, GSH, and (f) sodium 2,3-dimercapto-1-propanesulfonate,
DMPS [18, 19].
(a)(b)(c)(d)(e)(f)Studies into the mammalian metabolism of As(III) (Figure1(a))
have revealed a high propensity of this species to react with soft ligands,
such as the thiol group of cysteine [20–24].
Because the cysteine-containing tripeptide glutathione (GSH, Figure 1(e)) is
the most abundant endogenous thiol in mammalian hepatocyte cytoplasm (5 mM [25], whereas the concentration of
L-cysteine is 0.2–0.5 mM [26]),
the chemical reaction of As(III) with
three successive mole equivalents of GSH to arsenic triglutathione [As(SG)3], according to (1), is possibly the first step in
the hepatic metabolism of this oxyanion in vivo.(1)
Given that As(III) is known to be enzymatically methylated in the liver of most mammals [27–30], the detection of methylarsonous diglutathione [CH3As(SG)2]
in bile of As(III) treated rats [31–33] indicates that this metabolite is formed in the
liver, according to (2): (2)
In
view of the fact that DMA(III) has also
been identified in mammalian urine [12, 14, 15],
it is chemically feasible that dimethylarsinous glutathione [(CH3)2AsSG] is also formed in the liver, according
to (3):
(3)(CH3)2As-OH+GSH⇌(CH3)2As-SG+H2O.With respect to the biomethylation mechanism of As(III) in mammals, there are currently two proposed pathways  [34, 35]. The first one involves the enzymatic oxidative
methylation of As(III) to MMA(V),
which is enzymatically reduced to MMA(III),
and can then undergo a second enzymatic oxidative methylation reaction to DMA(V) [29, 36–38]. The alternative, more recently proposed scheme involves
the stepwise enzyme-mediated reductive methylation of As(SG)3 to CH3As(SG)2 and (CH3)2AsSG, using the same
methyl donor as in  scheme one—S-adenosyl-L-methionine [39, 40]. In this latter scheme, CH3As(SG)2 is then oxidized to MMA(V) by endogenously generated H2O2 while DMA(V) is produced in a similar manner from (CH3)2AsSG.
The entire biomethylation mechanism in hepatocytes, however, is not completely
understood because the concerted sequence of binding events of As(III) to endogenous
small-molecular-mass thiols [41] and the methylating enzymes remains unknown [34].Irrespective of
the mechanism of biomethylation, As(SG)3,
CH3As(SG)2, and (CH3)2AsSG likely play
important roles in the transport of methylated arsenicals from the liver to the
bloodstream [42–46]. Despite this, not much is known about the
stability of the As-S bonds in CH3As(SG)2 and (CH3)2AsSG
under conditions that resemble hepatocyte cytosol (phosphate
buffered saline, pH 7.4, 5.0 mM GSH, 37°C). Previously, the
formation of As(SG)3 from As(OH)3 and GSH has been
studied under simulated physiological conditions (phosphate
buffered saline, pH 7.4) by size-exclusion chromatography (SEC) [22]. This investigation revealed
that the on-column formation of the As(SG)3 complex strongly depends on
the GSH concentration in the mobile phase (5–7.5 mM favored)
and preferably occurs at pH 6.0–8.0. An increase of the column temperature from
4 to 37°C (at constant GSH concentration and at a mobile
phase pH of 7.4) resulted in retention shifts of the As(III) peaks toward the small-molecular-mass region, which
indicated that the As-S bonds in As(SG)3 are rather labile. This finding is
consistent with results attained in other studies  [20, 47–49].The mammalian metabolism of As(III) in hepatocytes is likely driven by its concerted
interactions with cytosolic GSH (5.0 mM) and proteins. In order to gain insight into the role that GSH plays in the
potential efflux of the generated As(III) metabolites from the liver into the systemic circulation, we have
chromatographed As(III), MMA(III), and DMA(III) under conditions that resemble the chemical conditions of mammalian hepatocyte
cytosol (phosphate buffered saline, 5–10 mM GSH, pH
7.4) in the absence of proteins. Investigations into the
temperature-dependent retention behavior of As(III),
MMA(III), and DMA(III) between 4 and 37°C provided insight into the formation (at
4°C) and the comparative stability (between
4 and 37°C) of the As-S bonds in the on-column formed complexes.
Our results constitute a first step toward better understanding the disposal of
As(III) metabolites from the liver to the bloodstream.


## 2. Experimental

Caution
Since inorganic and organic arsenicals are
established cytotoxins, genotoxins, and carcinogens [4, 50], measures must be implemented to reduce dermal and inhalatory
exposure. To this end, synthesis and solution preparation were conducted in a
glove box whilst wearing nitrile gloves and a respiratory mask.


### 2.1. Chemicals

Sodium hydroxide, phosphate buffered
saline (PBS) tablets, GSH, sodium
2,3-dimercapto-1-propanesulfonate (DMPS), blue dextran, oxidized glutathione (GSSG), and glycine (all>95%) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Sephadex G-15 (120μm mean spherical particles of dextran cross-linked
with epichlorohydrin, exclusion limit: molecular mass (MM) <1500 Da) was purchased from GE Healthcare.
Sodium arsenite (NaAsO2) was
obtained from GFS Chemicals (>99%). The
source of MMA(III) and DMA(III) was the solid methyldiiodoarsine (CH3AsI2) and the liquid
dimethyliodoarsine [(CH3)2AsI],
which were synthesized according to previously established procedures [51, 52]. To verify the purity of
these trivalent organoarsenicals, H1 NMR spectra were obtained for
these compounds (chemical shifts: 1.28 ppm for MMA(III) and 1.53 ppm for DMA(III)) and the
starting material (chemical shifts: 1.44 ppm for MMA(V) and 1.97 ppm for DMA(V)). The obtained chemical
shifts were found to be akin to those previously reported for these compounds [48, 53]. Arsenobetaine
bromide (AsB) was synthesized according
to a published procedure and its purity was verified by its melting point
(experimental: 225°C versus reported: 227°C) [54]. All solutions, including
mobile phases, were prepared with water from a simplicity water purification
system (resistivity 18.2 MΩ⋅cm, Millipore).


### 2.2. Solutions

To avoid
oxidation of GSH in aqueous mobile phases, all GSH-containing solutions were
prepared fresh prior to each chromatographic run and used within 4 hours. PBS-buffer was prepared by dissolving PBS tablets in the
appropriate volume of water. After the dissolution of a monothiol (5 or 10 mM GSH) and/or a dithiol (1 mM DMPS) in 400 mL PBS, the pH of the mobile phase was adjusted
to 7.4 with sodium hydroxide (4.0 M) using a VWR Symphony SB20 pH meter and filtered through a 0.45μm Nylon
membrane (Alltech).Aqueous
solutions of As(III), MMA(III), DMA(III),
and AsB were prepared by dissolving NaAsO2, CH3AsI2,
(CH3)2AsI, and AsB
in water to obtain a concentration of 10μg As in 20μL. It is generally
accepted that MMA(III) and DMA(III) are the only species that are formed upon
hydrolysis of CH3AsI2 and (CH3)2AsI
in water [18, 53]. Even though all
chromatograms were generated with the acidic solutions of MMA(III) and DMA(III) (hydroiodic acid is formed during the hydrolysis of CH3AsI2 and (CH3)2AsI in water), the retention
times (tr) of MMA(III) and DMA(III) were reduced by only∼20 seconds when neutralized solutions were injected. To
mitigate oxidation to the pentavalent state, As(III) and MMA(III) solutions were prepared
every 14 days while DMA(III) was
prepared fresh every 2-3 days [55–57]. All solutions were stored in
septum glass vials at 4°C.


### 2.3. Instrumentation

The liquid chromatographic (LC) system consisted of a Waters 510 high-performance LC isocratic dual-piston pump, a Rheodyne
six-port injection valve (20μL sample loop),
a glass thermostatable SEC column packed with Sephadex G-15 (31×1.0 cm, GE Healthcare), and a cellulose filter
on the column head (1.0 cm × 1.0 mm, GE Healthcare).
After thermostating the packed column at the desired temperature of 4, 25, or
37°C (NESLAB RTE-7 digital one refrigerated bath, Thermo Scientific), it was
equilibrated with at least 60 mL of mobile phase before the As compounds were
injected. A flow rate of 1.0 mL/min was used throughout this study and each
retention time was determined in triplicate (RSD <1.0%) after the individual injection of each arsenical.As-specific
detection was achieved with a Prodigy, high-dispersion, radial-view ICP-AES (Teledyne Leeman Labs, Hudson, NH, USA) by monitoring the As atomic
emission line at 189.042 nm. Hyphenation of the LC system to the ICP-AES was accomplished
by connecting the LC column exit to the concentric glass tube nebulizer with a
polyethylene tube (38 cm,
0.13 mm I.D.). The plasma Ar gas-flow rate and the nebulizer
gas pressure were 19 L/min and the
radiofrequency power output was 1.3 kW. Time scans
were performed using the time-resolved analysis mode (Salsa
software version 3.0) with a data acquisition rate of one data
point every 1.5 seconds. The data were exported and smoothed (bisquares weighting) using commercially available
software (SigmaPlot 9.0). The chromatographic window of the packed
Sephadex G-15 column was determined by injecting aqueous solutions of blue
dextran (MM 2 MDa,tr~6 minutes), to define the
exclusion volume (V0), and
glycine (MM 75 Da, tr~15 minutes), to define the
inclusion volume (Vi). The
column was size calibrated with aqueous solutions of
GSSG (MM∼600 Da, tr~10 minutes) and GSH (MM∼300 Da, tr~13 minutes). All calibration
experiments were performed with a PBS mobile phase (pH 7.4 at 25°C), while the
C atomic emission line at (193.091 nm) was monitored (Figure 2). Previous
studies have demonstrated that the pore size of Sephadex stationary phases does
not change appreciably in the 4–37°C temperature range
[22].Figure 2
Superimposed chromatograms of size calibration standards. Peaks from left to right represent blue dextran (MM 2 MDa), GSSG (MM∼600 Da), GSH (MM∼300 Da), and glycine (MM 75 Da). Stationary phase: Sephadex G-15 (31×1.0 cm I.D.); mobile phase: PBS-buffer (pH 7.4), flow rate: 1.0 mL/min; detector: ICP-AES at 193.091 nm; injection volume: 20μL.


## 2.1. Chemicals

Sodium hydroxide, phosphate buffered
saline (PBS) tablets, GSH, sodium
2,3-dimercapto-1-propanesulfonate (DMPS), blue dextran, oxidized glutathione (GSSG), and glycine (all>95%) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Sephadex G-15 (120μm mean spherical particles of dextran cross-linked
with epichlorohydrin, exclusion limit: molecular mass (MM) <1500 Da) was purchased from GE Healthcare.
Sodium arsenite (NaAsO2) was
obtained from GFS Chemicals (>99%). The
source of MMA(III) and DMA(III) was the solid methyldiiodoarsine (CH3AsI2) and the liquid
dimethyliodoarsine [(CH3)2AsI],
which were synthesized according to previously established procedures [51, 52]. To verify the purity of
these trivalent organoarsenicals, H1 NMR spectra were obtained for
these compounds (chemical shifts: 1.28 ppm for MMA(III) and 1.53 ppm for DMA(III)) and the
starting material (chemical shifts: 1.44 ppm for MMA(V) and 1.97 ppm for DMA(V)). The obtained chemical
shifts were found to be akin to those previously reported for these compounds [48, 53]. Arsenobetaine
bromide (AsB) was synthesized according
to a published procedure and its purity was verified by its melting point
(experimental: 225°C versus reported: 227°C) [54]. All solutions, including
mobile phases, were prepared with water from a simplicity water purification
system (resistivity 18.2 MΩ⋅cm, Millipore).


## 2.2. Solutions

To avoid
oxidation of GSH in aqueous mobile phases, all GSH-containing solutions were
prepared fresh prior to each chromatographic run and used within 4 hours. PBS-buffer was prepared by dissolving PBS tablets in the
appropriate volume of water. After the dissolution of a monothiol (5 or 10 mM GSH) and/or a dithiol (1 mM DMPS) in 400 mL PBS, the pH of the mobile phase was adjusted
to 7.4 with sodium hydroxide (4.0 M) using a VWR Symphony SB20 pH meter and filtered through a 0.45μm Nylon
membrane (Alltech).Aqueous
solutions of As(III), MMA(III), DMA(III),
and AsB were prepared by dissolving NaAsO2, CH3AsI2,
(CH3)2AsI, and AsB
in water to obtain a concentration of 10μg As in 20μL. It is generally
accepted that MMA(III) and DMA(III) are the only species that are formed upon
hydrolysis of CH3AsI2 and (CH3)2AsI
in water [18, 53]. Even though all
chromatograms were generated with the acidic solutions of MMA(III) and DMA(III) (hydroiodic acid is formed during the hydrolysis of CH3AsI2 and (CH3)2AsI in water), the retention
times (tr) of MMA(III) and DMA(III) were reduced by only∼20 seconds when neutralized solutions were injected. To
mitigate oxidation to the pentavalent state, As(III) and MMA(III) solutions were prepared
every 14 days while DMA(III) was
prepared fresh every 2-3 days [55–57]. All solutions were stored in
septum glass vials at 4°C.


## 2.3. Instrumentation

The liquid chromatographic (LC) system consisted of a Waters 510 high-performance LC isocratic dual-piston pump, a Rheodyne
six-port injection valve (20μL sample loop),
a glass thermostatable SEC column packed with Sephadex G-15 (31×1.0 cm, GE Healthcare), and a cellulose filter
on the column head (1.0 cm × 1.0 mm, GE Healthcare).
After thermostating the packed column at the desired temperature of 4, 25, or
37°C (NESLAB RTE-7 digital one refrigerated bath, Thermo Scientific), it was
equilibrated with at least 60 mL of mobile phase before the As compounds were
injected. A flow rate of 1.0 mL/min was used throughout this study and each
retention time was determined in triplicate (RSD <1.0%) after the individual injection of each arsenical.As-specific
detection was achieved with a Prodigy, high-dispersion, radial-view ICP-AES (Teledyne Leeman Labs, Hudson, NH, USA) by monitoring the As atomic
emission line at 189.042 nm. Hyphenation of the LC system to the ICP-AES was accomplished
by connecting the LC column exit to the concentric glass tube nebulizer with a
polyethylene tube (38 cm,
0.13 mm I.D.). The plasma Ar gas-flow rate and the nebulizer
gas pressure were 19 L/min and the
radiofrequency power output was 1.3 kW. Time scans
were performed using the time-resolved analysis mode (Salsa
software version 3.0) with a data acquisition rate of one data
point every 1.5 seconds. The data were exported and smoothed (bisquares weighting) using commercially available
software (SigmaPlot 9.0). The chromatographic window of the packed
Sephadex G-15 column was determined by injecting aqueous solutions of blue
dextran (MM 2 MDa,tr~6 minutes), to define the
exclusion volume (V0), and
glycine (MM 75 Da, tr~15 minutes), to define the
inclusion volume (Vi). The
column was size calibrated with aqueous solutions of
GSSG (MM∼600 Da, tr~10 minutes) and GSH (MM∼300 Da, tr~13 minutes). All calibration
experiments were performed with a PBS mobile phase (pH 7.4 at 25°C), while the
C atomic emission line at (193.091 nm) was monitored (Figure 2). Previous
studies have demonstrated that the pore size of Sephadex stationary phases does
not change appreciably in the 4–37°C temperature range
[22].Figure 2
Superimposed chromatograms of size calibration standards. Peaks from left to right represent blue dextran (MM 2 MDa), GSSG (MM∼600 Da), GSH (MM∼300 Da), and glycine (MM 75 Da). Stationary phase: Sephadex G-15 (31×1.0 cm I.D.); mobile phase: PBS-buffer (pH 7.4), flow rate: 1.0 mL/min; detector: ICP-AES at 193.091 nm; injection volume: 20μL.


## 3. Results and Discussion

Even
though the mammalian metabolism of As(III) is not entirely understood, still As(SG)3,
CH3As(SG)2, and (CH3)2AsSG likely play an important
role [32, 43, 45, 47]. Despite this, only the stability of the As-S bonds in As(SG)3 have been investigated under conditions that resemble mammalian hepatocyte cytosol [22]. This was established using the “retention analysis method” which was
originally developed to study the reversible oncolumn formation of drug-protein
complexes by LC [58, 59]. Employing this approach, we
studied the stability of trivalent As-(GS)x complexes (where x=1–3) by
independently chromatographing As(III),
MMA(III), and DMA(III) on an SEC column using GSH-containing PBS mobile phases (5
or 10 mM GSH, pH 7.4; Figure 3).Figure 3
The superimposed chromatograms of AsB, As(III), MMA  (III), and DMA(III) as a function of the SEC column temperature at 4°C (—), 25°C (- - -), or 37°C
(– – –). Stationary phase: Sephadex G-15 (31×1.0 cm
I.D.); mobile phase: PBS buffer containing 10 mM GSH adjusted to pH 7.4, flow rate: 1.0 mL/min; detector: ICP-AES at 189.042 nm; injection volume: 20μL (10μg As per compound).SEC was selected as the separation medium because previous studies
into the reaction of the aforementioned trivalent arsenicals with GSH revealed
the formation of As(SG)3 (MM∼900 Da), CH3As(SG)2 (MM∼600 Da), and
(CH3)2AsSG
(MM∼300 Da) [20, 23, 48, 49, 60], which all differ in their hydrodynamic radii. Therefore, the ideal
SEC stationary phase for the separation of the on-column formed complexes must
have an appropriate fractionation window. The most suited SEC stationary phase
was Sephadex G-15 since it offers an exclusion limit of <1500 Da [61]. Because of the particle size (120μm
diameter) and particle size distribution (60–180μm) of this stationary phase [61], however, relatively broad chromatographic peaks are expected, as has been previously
observed for As(SG)3 [22].


### 3.1. Temperature-Dependent On-Column Formation/Stability of Trivalent As-(GS)x Complexes

To determine the influence of temperature on the
on-column formation and stability of complexes that were formed between the injected trivalent As
compounds and the mobile phase thiol (10 mM GSH),
As(III), MMA(III), and DMA(III) were chromatographed
on a Sephadex G-15 column at 4, 25, and 37°C. An additional arsenical, AsB (Figure 1(d)), that does not interact with GSH, was also
chromatographed under these conditions as an internal standard. The observed
retention behavior of As(III),
MMA(III), DMA(III),
and AsB is depicted in Figure 3.In
general, all four arsenicals eluted within the chromatographic window. As
expected, AsB (MM∼178 Da) eluted in the small-molecular-mass region between GSH (MM∼300 Da) and glycine (MM 75 Da), irrespective of the column temperature (tr~14
minutes). In addition, the gradual reduction in peak width at baseline (wb) with an increase in temperature from 4
to 37°C can be rationalized by the
faster rate of diffusion of AsB into and out of the pores. Based on the
retention times of the peaks corresponding to As(III), MMA(III), and DMA(III) (tr range∼9–13.5 minutes) compared
to glycine (Vi, tr~15 minutes) and the fact that a
single chromatographic peak was obtained for each arsenical, they each must
have eluted from the column in the form of their respective (GS)x-complexes at all investigated
temperatures. This interpretation is further substantiated by the observation that
As(III), MMA(III),
and DMA(III) each eluted at∼20 minutes,
which is 5 minutes after the Vi, when PBS-containing mobile phases
without GSH were employed (data not shown).
An unspecified chemical interaction between these trivalent arsenicals and the
Sephadex G-15 matrix is most likely the cause of this behavior and has been
previously observed for As(III) on a
similar stationary phase material [22]. Presumably, the free hydroxyl groups of
the Sephadex G-15 matrix (stemming from the dextran
groups) interacted with the hydroxyl group(s) of the arsenicals via hydrogen bonding; hence, retarding their migration
through the column. It is chemically improbable that the observed retention
time changes between 4 and 37°C with the GSH-containing mobile phases were
caused by the aforementioned unspecified chemical interaction because all injected
arsenicals reacted first with GSH in the interstitial volume of the filter (∼80μL) prior to encountering the stationary phase pores.


### 3.2. Rationalization of the Chemical Structure of the On-Column Formed Complexes

The elution order of the injected trivalent arsenicals at 4°C was As(III) (tr~9 minutes), MMA(III) (tr~10 minutes), and
DMA(III) (tr~13 minutes), which
implies that the hydrodynamic
radii of the on-column formed trivalent As-(GS)x complexes decreased in this order. Based on the known chemical affinity of As(III), MMA(III), and DMA(III) for GSH [22, 23, 62]
(1)–(3), this order
of elution strongly indicates that As(SG)3,
CH3As(SG)2,
and (CH3)2AsSG had
formed on the column head.
Evidence in favor of the on-column formation of As(SG)3 (MM∼900 Da) comes from the
observation that As(III) eluted
between V0 and the MM 600 calibration standard (GSSG),
which indicates that a complex with an MM between 1500 and 600 Da had formed (Figure 3). Similarly, CH3As(SG)2 and (CH3)2AsSG were
most likely formed on the column head because the retention times corresponding
to MMA(III) and DMA(III) were identical to those of GSSG (MM∼600 Da) and GSH (MM∼300 Da; Figure 3, vertical-dashed lines). These trivalent
As-(GS)x complexes could not be
structurally characterized in the column effluent by electrospray ionization massspectrometry (ESI-MS) because the
temperature of the electrospray ion source chamber (120–365°C,
Esquire 3000 ESI-quadrupole ion trap mass spectrometer) would have
dissociated these thermally labile complexes (CH3As(SG)2 decomposes at 180°C, while (CH3)2AsSG
decomposes at 100°C [23]) during the ionization process, prior to mass analysis. Moreover,
as advised by the instrument supplier (Bruker Daltonics,
Billerica, MA, USA),
the salt concentration of mobile phase buffers that can be analyzed by ESI-MS
must be below 10 mM (total salt in the utilized PBS buffer
was∼164 mM). Nevertheless, the alignment of the on-column formed
complexes with the MM standards (Figure 3) in conjunction with the propensity of trivalent As compounds to react with soft
thiol ligands in aqueous solution to form As(SG)3,
CH3As(SG)2, and (CH3)2AsSG [20–24] strongly suggests that the latter species were formed on the
column.


### 3.3. Retention Behavior of As(III), MMA(III), and DMA(III)

With
regard to the retention behavior of As(III),
the peak that was observed at 4°C showed considerable tailing, which suggests
that As(SG)3 and an
additional complex with a smaller hydrodynamic radius,
possibly HOAs(SG)2, was
formed on the column. An increase of the column temperature from 4 to 37°C resulted
in a 4.6 minute retention shift of the As(III) peak toward the small-molecular-mass region (Figure 3),
which can be interpreted in two different ways. Firstly, the alignment of this
peak with GSSG at 25°C and with GSH at 37°C implies similar hydrodynamic radii,
which indicates that HOAs(SG)2 and (HO)2AsSG were likely formed
on the column under these conditions. Even though the peak corresponding to As(III) at 37°C did not
perfectly align with the MM 300 calibration standard (GSH),
it eluted closer to this standard than to glycine, which suggests that the As(III) was loosely bound to
one GS-moiety. Alternatively, the three GS-moieties that are bound to As(III) at 4°C could undergo
faster exchange upon increase of the temperature from 4 to 37°C (1). Nonetheless, these results strongly confirm the
previously demonstrated lability of the As-S bonds in As(SG)3 [20, 22, 47–49].The temperature-dependent retention behavior of MMA(III) and DMA(III) was significantly different from that of As(III) because the retention time of the peaks corresponding to MMA(III) and DMA(III) shifted only marginally (∼40 seconds for both versus 4.6 minutes for As(III)) upon
an increase in the column temperature from 4 to 37°C (Figure 3). Hence the on-column formation of the
corresponding trivalent As-(GS)x-complexes
(where x=1 or 2) was only minimally
affected by the column temperature. This, in turn, implies
that the As-S bonds in CH3As(SG)2 and (CH3)2AsSG are more stable than those in As(SG)3. Overall, these results, which were obtained under simulated
physiological conditions, are in excellent accord with previous observations,
which were conducted under nonphysiological conditions. In particular, these latter
studies revealed that CH3As(SG)2 and (CH3)2AsSG
could be synthesized in aqueous solution [23], whereas As(SG)3 could only be synthesized in alcoholic solutions (alcohol
apparently stabilizes the hydrolytically labile As-S bonds) [20, 49].The
entire temperature-dependent retention behavior of As(III), MMA(III), DMA(III), and
AsB was repeated with a 5 mM GSH-containing mobile phase (PBS,
pH 7.4) on the same SEC column. These results were identical to
those illustrated in Figure3 (data not shown), which makes the observed comparative stability of the on-column formed
complexes relevant to mammalian, protein-free, hepatocyte cytosol.


### 3.4. Biochemical Ramifications of the Obtained Results

ATP-driven GS-X conjugate export pumps,
which shuttle xenobiotic-GS-conjugates across phospholipid bilayer membranes via multidrug resistance proteins 1
and 2, are known to exist at the basolateral and apical hepatocyte membrane,
respectively [42–45]. Therefore, our findingsraise the possibility that the biomethylation of As(III) to MMA(III) and DMA(III) in the liver of mammals may have
evolved simply to export As(III) into the bloodstream for subsequent
urinary excretion via the kidney. This rather simplistic hypothesis could
explain why DMA(III) has been detected
in rat erythrocytes bound to hemoglobin [63] and why MMA(III) and DMA(III) have been detected in mammalian urine [12–16]. The exact mechanism by which these
trivalent arsenicals are exported from the liver to the bloodstream, however, needs to be further investigated.


### 3.5. Influence of DMPS Addition to the Mobile Phase on Complex Formation at 37°C

To substantiate that the retention shift of As(III) to a smaller retention time upon the addition of GSH to themobile phase was
caused by the on-column formation of As(SG)3,
a mobile phase containing both GSH (5 mM) and DMPS (1 mM, Figure 1(f)), a chelating agent that forms a stronger complex
with As(III) than GSH, was investigated. This resulted in the expected increase in the retention time of As(III) to Vi(tr≈17 minutes), which
is inaccord with previous findings [22]. In contrast, no As
peaks for MMA(III) and DMA(III) were detected in the column
effluent when a PBS mobile phase containing GSH (5 mM) and DMPS (1 mM) was employed. The cause
of this reproducible behavior is not presently understood.


## 3.1. Temperature-Dependent On-Column Formation/Stability of Trivalent As-(GS)x Complexes

To determine the influence of temperature on the
on-column formation and stability of complexes that were formed between the injected trivalent As
compounds and the mobile phase thiol (10 mM GSH),
As(III), MMA(III), and DMA(III) were chromatographed
on a Sephadex G-15 column at 4, 25, and 37°C. An additional arsenical, AsB (Figure 1(d)), that does not interact with GSH, was also
chromatographed under these conditions as an internal standard. The observed
retention behavior of As(III),
MMA(III), DMA(III),
and AsB is depicted in Figure 3.In
general, all four arsenicals eluted within the chromatographic window. As
expected, AsB (MM∼178 Da) eluted in the small-molecular-mass region between GSH (MM∼300 Da) and glycine (MM 75 Da), irrespective of the column temperature (tr~14
minutes). In addition, the gradual reduction in peak width at baseline (wb) with an increase in temperature from 4
to 37°C can be rationalized by the
faster rate of diffusion of AsB into and out of the pores. Based on the
retention times of the peaks corresponding to As(III), MMA(III), and DMA(III) (tr range∼9–13.5 minutes) compared
to glycine (Vi, tr~15 minutes) and the fact that a
single chromatographic peak was obtained for each arsenical, they each must
have eluted from the column in the form of their respective (GS)x-complexes at all investigated
temperatures. This interpretation is further substantiated by the observation that
As(III), MMA(III),
and DMA(III) each eluted at∼20 minutes,
which is 5 minutes after the Vi, when PBS-containing mobile phases
without GSH were employed (data not shown).
An unspecified chemical interaction between these trivalent arsenicals and the
Sephadex G-15 matrix is most likely the cause of this behavior and has been
previously observed for As(III) on a
similar stationary phase material [22]. Presumably, the free hydroxyl groups of
the Sephadex G-15 matrix (stemming from the dextran
groups) interacted with the hydroxyl group(s) of the arsenicals via hydrogen bonding; hence, retarding their migration
through the column. It is chemically improbable that the observed retention
time changes between 4 and 37°C with the GSH-containing mobile phases were
caused by the aforementioned unspecified chemical interaction because all injected
arsenicals reacted first with GSH in the interstitial volume of the filter (∼80μL) prior to encountering the stationary phase pores.


## 3.2. Rationalization of the Chemical Structure of the On-Column Formed Complexes

The elution order of the injected trivalent arsenicals at 4°C was As(III) (tr~9 minutes), MMA(III) (tr~10 minutes), and
DMA(III) (tr~13 minutes), which
implies that the hydrodynamic
radii of the on-column formed trivalent As-(GS)x complexes decreased in this order. Based on the known chemical affinity of As(III), MMA(III), and DMA(III) for GSH [22, 23, 62]
(1)–(3), this order
of elution strongly indicates that As(SG)3,
CH3As(SG)2,
and (CH3)2AsSG had
formed on the column head.
Evidence in favor of the on-column formation of As(SG)3 (MM∼900 Da) comes from the
observation that As(III) eluted
between V0 and the MM 600 calibration standard (GSSG),
which indicates that a complex with an MM between 1500 and 600 Da had formed (Figure 3). Similarly, CH3As(SG)2 and (CH3)2AsSG were
most likely formed on the column head because the retention times corresponding
to MMA(III) and DMA(III) were identical to those of GSSG (MM∼600 Da) and GSH (MM∼300 Da; Figure 3, vertical-dashed lines). These trivalent
As-(GS)x complexes could not be
structurally characterized in the column effluent by electrospray ionization massspectrometry (ESI-MS) because the
temperature of the electrospray ion source chamber (120–365°C,
Esquire 3000 ESI-quadrupole ion trap mass spectrometer) would have
dissociated these thermally labile complexes (CH3As(SG)2 decomposes at 180°C, while (CH3)2AsSG
decomposes at 100°C [23]) during the ionization process, prior to mass analysis. Moreover,
as advised by the instrument supplier (Bruker Daltonics,
Billerica, MA, USA),
the salt concentration of mobile phase buffers that can be analyzed by ESI-MS
must be below 10 mM (total salt in the utilized PBS buffer
was∼164 mM). Nevertheless, the alignment of the on-column formed
complexes with the MM standards (Figure 3) in conjunction with the propensity of trivalent As compounds to react with soft
thiol ligands in aqueous solution to form As(SG)3,
CH3As(SG)2, and (CH3)2AsSG [20–24] strongly suggests that the latter species were formed on the
column.


## 3.3. Retention Behavior of As(III), MMA(III), and DMA(III)

With
regard to the retention behavior of As(III),
the peak that was observed at 4°C showed considerable tailing, which suggests
that As(SG)3 and an
additional complex with a smaller hydrodynamic radius,
possibly HOAs(SG)2, was
formed on the column. An increase of the column temperature from 4 to 37°C resulted
in a 4.6 minute retention shift of the As(III) peak toward the small-molecular-mass region (Figure 3),
which can be interpreted in two different ways. Firstly, the alignment of this
peak with GSSG at 25°C and with GSH at 37°C implies similar hydrodynamic radii,
which indicates that HOAs(SG)2 and (HO)2AsSG were likely formed
on the column under these conditions. Even though the peak corresponding to As(III) at 37°C did not
perfectly align with the MM 300 calibration standard (GSH),
it eluted closer to this standard than to glycine, which suggests that the As(III) was loosely bound to
one GS-moiety. Alternatively, the three GS-moieties that are bound to As(III) at 4°C could undergo
faster exchange upon increase of the temperature from 4 to 37°C (1). Nonetheless, these results strongly confirm the
previously demonstrated lability of the As-S bonds in As(SG)3 [20, 22, 47–49].The temperature-dependent retention behavior of MMA(III) and DMA(III) was significantly different from that of As(III) because the retention time of the peaks corresponding to MMA(III) and DMA(III) shifted only marginally (∼40 seconds for both versus 4.6 minutes for As(III)) upon
an increase in the column temperature from 4 to 37°C (Figure 3). Hence the on-column formation of the
corresponding trivalent As-(GS)x-complexes
(where x=1 or 2) was only minimally
affected by the column temperature. This, in turn, implies
that the As-S bonds in CH3As(SG)2 and (CH3)2AsSG are more stable than those in As(SG)3. Overall, these results, which were obtained under simulated
physiological conditions, are in excellent accord with previous observations,
which were conducted under nonphysiological conditions. In particular, these latter
studies revealed that CH3As(SG)2 and (CH3)2AsSG
could be synthesized in aqueous solution [23], whereas As(SG)3 could only be synthesized in alcoholic solutions (alcohol
apparently stabilizes the hydrolytically labile As-S bonds) [20, 49].The
entire temperature-dependent retention behavior of As(III), MMA(III), DMA(III), and
AsB was repeated with a 5 mM GSH-containing mobile phase (PBS,
pH 7.4) on the same SEC column. These results were identical to
those illustrated in Figure3 (data not shown), which makes the observed comparative stability of the on-column formed
complexes relevant to mammalian, protein-free, hepatocyte cytosol.


## 3.4. Biochemical Ramifications of the Obtained Results

ATP-driven GS-X conjugate export pumps,
which shuttle xenobiotic-GS-conjugates across phospholipid bilayer membranes via multidrug resistance proteins 1
and 2, are known to exist at the basolateral and apical hepatocyte membrane,
respectively [42–45]. Therefore, our findingsraise the possibility that the biomethylation of As(III) to MMA(III) and DMA(III) in the liver of mammals may have
evolved simply to export As(III) into the bloodstream for subsequent
urinary excretion via the kidney. This rather simplistic hypothesis could
explain why DMA(III) has been detected
in rat erythrocytes bound to hemoglobin [63] and why MMA(III) and DMA(III) have been detected in mammalian urine [12–16]. The exact mechanism by which these
trivalent arsenicals are exported from the liver to the bloodstream, however, needs to be further investigated.


## 3.5. Influence of DMPS Addition to the Mobile Phase on Complex Formation at 37°C

To substantiate that the retention shift of As(III) to a smaller retention time upon the addition of GSH to themobile phase was
caused by the on-column formation of As(SG)3,
a mobile phase containing both GSH (5 mM) and DMPS (1 mM, Figure 1(f)), a chelating agent that forms a stronger complex
with As(III) than GSH, was investigated. This resulted in the expected increase in the retention time of As(III) to Vi(tr≈17 minutes), which
is inaccord with previous findings [22]. In contrast, no As
peaks for MMA(III) and DMA(III) were detected in the column
effluent when a PBS mobile phase containing GSH (5 mM) and DMPS (1 mM) was employed. The cause
of this reproducible behavior is not presently understood.


## 4. Conclusion

The SEC-based “retention analysis method” approach in conjunction with an As-specific detector was employed to study the comparative on-column formation of trivalent As-(GS)x-complexes.
This was achieved by using mobile phases resembling the
chemical composition of mammalian, protein-free hepatocyte cytosol (5 or 10 mM GSH, PBS, pH 7.4) at column temperatures of 4, 25, or 37°C. The separate
injections of As(III), MMA(III), and DMA(III) and their observed retention behavior provided evidence for the on-column formation
of more stable As-S bonds in CH3As(SG)2 and (CH3)2AsSG than
in As(SG)3. These findings imply that the stability of As-S bonds could be critically involved in the
disposition/excretion of methylated trivalent As compounds in mammals. Future
investigations should be aimed at identifying whether CH3As(SG)2 and (CH3)2AsSG
are in fact translocated across the hepatocyte membrane into the bloodstream in vivo.



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*Source: 539082-2008-05-15.xml*