Animal species difference in the uptake of dimethylarsinous acid (DMAIII) by red blood cells

Article abstract of DOI:10.1021/tx015537k

The animal species difference in the metabolism of arsenic was studied from the viewpoint of the mechanism underlying its distribution in the form of dimethylated arsenic in red blood cells (RBCs). Dimethylarsinic (DMA^V) and dimethylarsinous (DMA^III) acids were incubated with rat, hamster, mouse, and human RBCs, and the uptake rates and chemical forms of arsenic were determined. Although DMA^V was practically not or taken up slowly by RBCs of all the present animal species, DMA^III was taken efficiently in the order of rat > hamster > human, RBCs of mice taking it up less efficiently and with a different pattern from the former three animals. Further, although DMA^III taken up by rat RBCs was retained, that by hamster ones was effluxed in the form of DMA^V. The uptake of DMA^III and efflux of DMA^V took place much more slowly in human RBCs than rat and hamster ones. The uptake of DMA^III by RBCs was inhibited on the oxidation of glutathione with diamide. Incubation of DMA^III, but not of DMA^V, with a hemolysate produced a high molecular weight complex, which increases in the presence of glutathione, suggesting that DMA^III taken up by RBCs is retained through the formation of a complex with protein(s) specific to animal species, and effluxed from RBCs after being oxidized to DMA^V. These results indicate that DMA is taken up by RBCs in the form of DMA^III, and that the uptake and efflux rates are dependent on the animal species, the effluxed arsenic being DMA^V. The present results suggest that the uptake of DMA by RBCs is an additional contributing factor to the animal species difference in the metabolism of arsenic in addition to the reduction and methylation capacity in the liver.

Full text of DOI:10.1021/tx015537k

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446
Chem. Res. Toxicol. 2001, 14, 1446-1452
An im a l Sp ecies Differ en ce in th e Up ta k e of
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Dim eth yla r sin ou s Acid (DMA ) by Red Blood Cells
Yamato Shiobara, Yasumitsu Ogra, and Kazuo T. Suzuki*
Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, J apan
Received J uly 5, 2001
The animal species difference in the metabolism of arsenic was studied from the viewpoint
of the mechanism underlying its distribution in the form of dimethylated arsenic in red blood
V
III
cells (RBCs). Dimethylarsinic (DMA ) and dimethylarsinous (DMA ) acids were incubated
with rat, hamster, mouse, and human RBCs, and the uptake rates and chemical forms of arsenic
V
were determined. Although DMA was practically not or taken up slowly by RBCs of all the
present animal species, DMA was taken efficiently in the order of rat > hamster > human,
RBCs of mice taking it up less efficiently and with a different pattern from the former three
animals. Further, although DMA taken up by rat RBCs was retained, that by hamster ones
was effluxed in the form of DMA . The uptake of DMA and efflux of DMA took place much
more slowly in human RBCs than rat and hamster ones. The uptake of DMA by RBCs was
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inhibited on the oxidation of glutathione with diamide. Incubation of DMA , but not of DMA ,
with a hemolysate produced a high molecular weight complex, which increases in the presence
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of glutathione, suggesting that DMA taken up by RBCs is retained through the formation of
a complex with protein(s) specific to animal species, and effluxed from RBCs after being oxidized
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to DMA . These results indicate that DMA is taken up by RBCs in the form of DMA , and
that the uptake and efflux rates are dependent on the animal species, the effluxed arsenic
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being DMA . The present results suggest that the uptake of DMA by RBCs is an additional
contributing factor to the animal species difference in the metabolism of arsenic in addition to
the reduction and methylation capacity in the liver.
In tr od u ction
Arsenic has long been known to cause the toxicity in
Sch em e 1. Red u ction a n d Meth yla tion P a th w a ys
for Ar sen ic
humans (1), and has been correlated convincingly with
cancers of the skin, lungs, liver, kidneys, and bladder (2-
5
). It has been generally believed that reduction and
methylation of inorganic arsenic comprise detoxification
mechanisms (3, 6-9). The toxicity of arsenic is known to
be manifested differently depending on the animal spe-
cies, owing to differences in the metabolic pathways (3,
1
0-13). The rat is one of the most tolerant species among
III
animal species (10), and arsenite (iAs ) is effectively
transformed to dimethylated arsenic (DMA), and then
the arsenic (possibly in the form of DMA) is mostly
distributed to red blood cells (RBCs) (14, 15), which we
also confirmed in a separate experiment (data not
shown). On the other hand, the hamster is one of the
animal species most sensitive to arsenic, and arsenite is
methylated less efficiently than in rats and distributed
to RBCs in only small quantities (16-18).
uptake of DMA by RBCs may differ among animal
species and be related with the difference in manifesta-
tion of the toxicity of arsenic among animal species.
Inorganic arsenic has been proposed to be metabolized
in the body through successive reduction and methylation
reactions leading to the major urinary metabolite DMA,
as illustrated in Scheme 1. Arsenite injected intrave-
nously into rats was not taken up or at least not
effectively taken up by RBCs, but was taken up by the
liver, and then the arsenic accumulated in the RBCs in
our preliminary experiment (data not shown). Further,
the methylation reaction seems to be not or less possible
in RBCs than in liver. Therefore, arsenic is assumed to
Arsenite is known to be taken up by and methylated
in the liver, and then DMA is distributed possibly in the
form of DMA to RBCs in rats, but not in hamsters (16-
1
8). These observations suggest that the reduction and
methylation capacity of the liver and/or the process of
*
To whom correspondence should be addressed. Fax/Phone: 81-
4
3-290-2891. E-mail: ktsuzuki@p.chiba-u.ac.jp.
1
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Abbreviations: iAs , arsenite; iAs , arsenate; DMA, dimethylated
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arsenic; DMA , dimethylarsinic acid; DMA , dimethylarsinous acid;
RBCs, red blood cells; diamide, azodicarboxylic acid bis(dimethyla-
mide); DIDS, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid disodium
salt; GSH, glutathione.
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0.1021/tx015537k CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/01/2001

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Difference in Arsenic (DMA ) Uptake by RBCs
Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1447
be effluxed in the form of DMA from the liver into the
bloodstream and then taken up by RBCs.
Up ta k e of Ar sen ic Com p ou n d s by Red Blood Cells. Male
Wistar rats and ICR mice, and Syrian hamsters were purchased
at 8 weeks of age from Clea J apan Co., Tokyo, J apan and J apan
SLC Inc., Hamamatsu, J apan, respectively. The animals were
fed a commercial diet (CE-2; Clea J apan Co.) and tap water ad
libitum.
Although it has been documented that the reduction
and methylation capacity is different among animal
species (3, 6-9), it is not known whether there is any
difference in the uptake of DMA among RBCs of different
animal species. It is also not known why arsenic ac-
cumulates preferentially in RBCs in rats (16-18). There-
fore, in the present study, we examined in vitro the
difference in the uptake of DMA by RBCs.
RBCs of each animal species were prepared from heparinized
blood, and were separated from plasma and a buffy coat by
centrifuging at 1600g for 15 min. The separated RBCs were re-
suspended in TBS, and then centrifuged to remove residual
plasma, only erythrocytes being detected under microscopic
examination.
DMAIII or DMAV was added to heparinized whole blood of
Wistar rats or to suspensions (10% in TBS) of rat, hamster,
mouse, and human RBCs at the concentration of 15 µg/mL
incubation solution, followed by incubation at 37 °C for up to 4
h. RBCs were removed by centrifugation at 8000g for 10 s, and
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DMA can be in two forms, dimethylarsinous (DMA )
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and dimethylarsinic (DMA ) acids, the latter form being
proposed to be the major urinary metabolite. However,
the former form was shown to be present as a urinary
metabolite in humans together with the latter form (19-
2
2), suggesting that DMA may be effluxed from liver to
then after ashing with HNO
arsenic in the medium was determined with an ICP MS (HP
500; Yokogawa Analytical Systems Co., Musashino, J apan).
3 2 2
and H O , the concentration of
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bloodstream in both forms. It should be noted that DMA
and MMA have been detected in the liver of hamsters
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4
(23).
The chemical form of arsenic in the medium was determined
with a cation exchange column (RSpak NN 614, 6.0 × 150 mm;
Showa Denko, Tokyo) by the HPLC-ICP MS method (22).
To confirm the uptake but not the adsorption of DMAIII on
In the present study, we examined whether there is
any difference in the uptake of DMA among RBCs of
different animal species and also difference in the chemi-
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cal species of DMA between DMA and DMA . RBCs
were prepared from three animal species, i.e., rats, mice,
and hamsters of different arsenic metabolisms (10), and
the outer membrane of RBCs, DMA was incubated in a 10%
suspension of rat RBCs or in a 10% suspension of the membrane
fraction separated from rat RBCs for 1 h under the same
conditions as mentioned above. The concentrations of arsenic
in the incubation media and the membrane fractions were
determined with an ICP MS. Arsenic was recovered in the RBCs
at 80.6% of the dose, i.e., 78.8% in the lysate, 0.97% in the
membrane fraction, and 0.82% in the precipitate. On the other
hand, when DMAV was incubated in a 10% suspension of the
membrane fraction, arsenic was recovered mostly (95.8% of the
dose) in the incubation medium without being precipitated with
the membrane fraction.
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also from humans. DMA was prepared from DMA in
our laboratory (22, 24).
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The present study revealed that only DMA , i.e., not
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DMA , is taken up efficiently by RBCs, and further the
uptake is dependent on the animal species, the uptake
by rat RBCs being most effective. These results suggest
that the species difference in the metabolism of arsenic,
and hence its toxicity, can be attributed not only to the
reduction and methylation capacity leading to DMA in
Suspensions (10% RBCs in TBS) of rat, hamster, mouse, and
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the liver but also to the reduction capacity of DMA to
human RBCs were incubated with different concentrations of
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diamide for 20 min at 37 °C, and then incubated with DMAIII
DMA in the liver followed by the uptake/retention
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and DMAV for 1 h at 37 °C.
capacity for DMA of RBCs. Further, the mechanism
Rat RBCs were lysed by freezing and thawing, and the
resulting lysate was centrifuged at 15000g for 20 min to obtain
a supernatant fraction. DMA and DMAV were incubated in
the supernatant fraction in the presence or absence of 1 mM
GSH at 37 °C for 1 h. The distribution of arsenic was determined
on a gel filtration column (Asahipak GS 220 HQ, 7.6 × 250 mm;
Showa Denko) by the HPLC-ICP MS method.
underlying the uptake/retention of DMA by RBCs was
examined.
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Exp er im en ta l P r oced u r es
Ca u tion . Inorganic arsenic compounds have been established
as human carcinogens (3). Ingestion of inorganic arsenic may
cause cancer of the skin, urinary bladder, kidney, lung, and liver
as well as with disorders of the circulatory and nervous system.
Deter m in a tion of Ar sen ic Con cen tr a tion s a n d Th eir
Ch em ica l F or m s. The concentrations of arsenic in whole blood,
plasma and medium were determined after wet-ashing with
V
Rea gen ts. Sodium dimethlyarsinite (DMA ), sodium thio-
3 2 2
HNO and H O , at m/z 75, with an ICP-MS.
sulfate, sodium disulfite, sulfuric acid, and nitric acid of reagent
grade were purchased from Wako Pure Chemicals Industries
Ltd., Osaka, J apan. Trizma Hydrochloride, Trizma Base and
azodicarboxylic acid bis(dimethylamide) (diamide) were pur-
chased from Sigma, St. Louis, MO. 4,4′-Diisothiocyano-2,2′-
stilbene disulfonic acid disodium salt (DIDS) was purchased
from ICN Pharmaceuticals, Inc., Costa Mesa, CA. The arsenic
standard solution (1000 ppm) used for ICP MS was purchased
from SPEX CentiPrep, Metuchen, NJ .
A 0.02 mL aliquot of plasma or medium containing an arsenic
compound was applied to a cation exchange column (NN-614),
and then the column was eluted with 36 mM formic acid/2 mM
ammonium formate buffer, pH 2.81, at 25 °C, with an HPLC
(PU 610; GL Sciences Co., Tokyo) at the flow rate of 0.6 mL/
min. The eluent was introduced directly into the nebulizer tube
of the ICP-MS to detect arsenic (m/z 75).
DMAIII was prepared from DMA according to the method of
Reay and Asher (24). Briefly, 0.28 g of sodium disulfite was
dissolved in 15 mL of 50 mM Tris-HCl buffered saline (TBS,
pH 7.4), and then 2 mL of 1% sodium thiosulfate was added to
the solution. After the addition of 0.1 mL of concentrated sulfuric
acid to the mixture, 1 vol of it was mixed with one volume of a
DMAV solution (2 mM), followed by incubation at room tem-
perature for 2 h. This reaction mixture was subsequently diluted
with the mobile phase for HPLC to the final concentration of
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Resu lts
Arsenic administered orally to rats in the form of
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arsenite (As ) was efficiently taken up by and ac-
cumulated in RBCs (data not shown), in the form of
dimethylated As (DMA) as also reported in the literature
(14, 15). Contrary to the accumulation of DMA in RBCs
in vivo, commercially available DMA was taken up only
slowly and marginally in vitro by the RBCs in heparin-
ized whole blood of rats, as shown in Figure 1. With
higher and lower concentrations of arsenic than the
5
-20 nM and then analyzed by the HPLC-ICP MS (mass
spectrometry with ionization by inductively coupled argon
plasma) method.

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448 Chem. Res. Toxicol., Vol. 14, No. 10, 2001
Shiobara et al.
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F igu r e 1. Uptake of dimethylarsinic acid (DMA ) by red blood
cells (RBCs) of rats and the effect of medium glucose on the
uptake. DMAV was incubated in heparinized blood of Wistar
rats at a concentration of 15 µg/mL blood at 37 °C for up to 8 h
in the presence of glucose at doses of 0, 5, 20, and 40 g/L. The
dotted horizontal line shows the concentration of arsenic
distributed evenly in the plasma and RBCs.
dotted line, arsenic was not or less efficiently taken up
and effectively taken up by RBCs, respectively.
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F igu r e 2. Chemical reduction of dimethylarsinic (DMA ) to
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III
dimethylarsinous (DMA ) acids, and uptake of DMA by rat
RBCs. (A) The distributions of arsenic on a cation exchange
column with the HPLC-ICP MS method were determined for
Although the concentration of arsenic in the plasma
decreased marginally and very slowly in vitro in whole
blood, the decrease was enhanced in the presence of
glucose depending on its concentration, as shown in
Figure 1. Nevertheless, the uptake of arsenic was limited
and slow compared with that in vivo (arsenic accumulates
in RBCs within several hours after an intravenous
injection of arsenite into rats, unpublished observation)
and it seems to be too slow for uptake by RBCs, being
rather filtered out by the glomerulus.
V
III
commercially available DMA and its reduced form, DMA . (B)
DMA or DMAV was incubated in heparinized blood of rat
origin, and the concentration of arsenic was plotted against the
incubation period. The dotted horizontal line shows the concen-
tration of arsenic distributed evenly in the plasma and RBCs.
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DMA is present in two forms, DMA and DMA , the
former being a commercially available form. The DMA
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used in the experiment in Figure 1 was DMA , which
may explain the slow and limited uptake. Therefore, the
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uptake of DMA in the reduced form, DMA , was exam-
ined, as shown in Figure 2. Commercially available
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DMA was reduced to DMA according to the reported
procedure (22, 24), as shown in Figure 2A. The two forms
of DMA were incubated in heparinized blood, as shown
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in Figure 2B. The reduced DMA (DMA ) was taken up
by RBCs within 10 min, which suggests that DMA is
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secreted from the liver in the form of DMA and then
taken up efficiently by RBCs. This also suggests that
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DMA is taken up much less and slowly by RBCs than
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DMA and hence is filtered out by the glomerulus rather
than being taken up by RBCs in vivo.
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F igu r e 3. Species differences in the uptake of dimethylarsinous
Although DMA was added to heparinized whole blood
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acid (DMA ) by rat, hamster, mouse and human RBCs in vitro.
at the concentration of 15 µg of As/mL whole blood, the
arsenic concentration was already lowered than the
calculated level of 21.2 µg/mL plasma (hematocrit value,
DMAIII or DMAV was incubated with suspended RBCs (10%
RBCs in Tris-HCl buffered saline) of rat (A), hamster (B), mouse
(C), and human (D) origin, and the concentration of arsenic was
plotted against the incubation period. The dotted horizontal line
shows the concentration of arsenic distributed evenly in the
medium and RBCs.
4
0.7%) immediately after the incubation and then in-
creased to the calculated level 10 min after the incubation
Figure 2). This rapid change suggests the homogeneous
(
distribution of arsenic between RBCs and plasma, fol-
lowed by the relocalization in the plasma. However, it
was not detected in a suspension of rat RBCs, as shown
in Figure 3A.
uptake of DMAIII by rat, hamster, mouse, and human
RBCs, as shown in Figure 3. As DMA was effectively
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taken up by RBCs without the presence of plasma
constituents, DMAIII and DMAV were incubated with
The results in Figure 2 indicate that the effective
uptake of DMA by RBCs in vivo can be attributed either
RBC suspensions in Tris-HCl buffered saline instead of
heparinized whole blood in the present experiment. It
was also confirmed that the decrease of arsenic in the
suspension medium of RBCs is due to the uptake of DMA
by RBCs but not due to the adsorption on the outer
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to the effective reduction of DMA to DMA in the liver
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or the effective uptake of DMA by RBCs. Then, the
latter possibility was examined in vitro by comparing the

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Difference in Arsenic (DMA ) Uptake by RBCs
Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1449
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F igu r e 4. Changes in the chemical form of arsenic in the medium on incubation of DMA with rat, hamster, and human RBCs.
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DMA was incubated with suspended RBCs (10% RBCs in Tris-HCl buffered saline) of rat (A), hamster (B), and human (C) origin,
or without RBCs in Tris-HCl buffered saline (D), at 37 °C for up to 4 h, and then the distribution of arsenic in the medium was
determined on a cation exchange column by the HPLC-ICP MS method. The vertical bar indicates the detection level for arsenic.
DMAIII can be oxidized spontaneously to DMA , this
oxidation was not observed under the present conditions
without RBCs, as shown in Figure 4D.
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membrane of RBCs, as described in the Experimental
Procedures.
DMA was taken up by RBCs at different rates
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depending on the animal species, the uptake by the rat
RBCs being the most efficient, followed by hamster,
The results shown in Figure 4 suggest that the species
difference in the uptake of DMA by RBCs is not due to
the difference in the channel responsible to the transport
through the membrane. Nevertheless, the responsible
channel seems to be important as to understanding the
mechanism underlying the species difference in the
metabolism of arsenic. The anion exchange carrier (band
3 protein) is the major integral protein of RBCs and
catalyses the passage of anions across the membrane (25,
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mouse and human ones. On the other hand, DMA was
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taken up less efficiently than DMA by the RBCs of all
animal species, being distributed homogeneously between
the RBCs and medium.
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DMA was shown to be taken up differently by RBCs
depending on the animal species, as shown in Figure 3.
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However, there is an alternative explanation; DMA may
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be taken up and retained in rat RBCs, while it may be
taken up but not retained in hamster, mouse, and human
RBCs. Therefore, the chemical form of arsenic in the
medium was determined by the HPLC-ICP MS method,
as shown in Figure 4.
26). The possibility of the transport of DMA through
the common channel for phosphate, carbonate, and
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sulfate was examined as to the uptake of DMA by
adding phosphate to suspensions of rat and hamster
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RBCs. Phosphate did not affect the uptake of DMA up
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DMA was effectively taken up by rat RBCs, while
to 5 mM (data not shown). Further, a specific inhibitor
of band 3 protein, DIDS (27), was not effective under the
conditions under which the transport of selenite into
RBCs is effectively inhibited (28), as shown in Figure 5;
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the intensity of DMA peak did not change with incuba-
tion time, as shown in Figure 4A. On the other hand, in
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the case of hamster RBCs, DMA decreased with time
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(
though less efficiently than that of rat ones), as shown
the uptake and oxidation of DMA by rat RBCs and also
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in Figure 4B, at the same time, the intensity of the DMA
peak increased with the incubation time and became
greater than that of the DMAIII peak after 1 h. The
results in Figure 4, panels A and B, suggest that, in rats,
DMA is secreted from the liver into the bloodstream in
the form of DMA and then taken up by and retained
in the RBCs. However, in hamsters, DMA is taken up
by RBCs and oxidized to DMA and then effluxed from
by hamster RBCs did not change in the presence of 50
µM DIDS, as shown in Figure 5, panels A, B and C, D,
respectively.
The animal species difference in the uptake of DMAIII
by RBCs is, thus, assumed to be dependent on the
difference in the oxidation/retention of arsenic in RBCs.
Since trivalent arsenic compounds are known to conju-
gate with glutathione (GSH) (29, 30), the effect of GSH
depletion on the uptake/retention of arsenic in RBCs was
examined in the presence of GSH-depleter diamide in the
RBC suspension, as shown in Figure 6.
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the RBCs without retaining them.
The chemical forms of arsenic in the medium were also
III
determined after incubation of DMA with human RBCs,
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as shown in Figure 4C. The DMA peak decreased less
DMA and DMA were incubated with rat RBCs in
the presence of different concentrations of the oxidizing
agent for GSH, diamide (31), for 1 h. The uptake of
slowly for human RBCs than for hamster ones, and the
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DMA peak increased much more slowly than in the case
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of hamsters, suggesting that the oxidation and efflux are
less efficient in human than in rat RBCs. Although
DMA decreased depending on the dose of diamide, while
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the concentration of DMA in the medium did not show

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450 Chem. Res. Toxicol., Vol. 14, No. 10, 2001
Shiobara et al.
F igu r e 5. Effect of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on the uptake of dimethylarsinous acid (DMAIII) on
III
incubation with rat and hamster RBCs. DMA was incubated with suspended RBCs (10% RBCs in Tris-HCl buffered saline) of rat
(
A, B) and hamster (C, D) origin in the presence (B, D) and absence (A, C) of 50 µM DIDS at 37 °C for up to 4 h, and then the
distribution of arsenic in the medium was determined on a cation exchange column by the HPLC-ICP MS method. The vertical bar
indicates the detection level for arsenic.
the DMAV peak did not increase with rat RBCs even
under more oxidized conditions with depletion of GSH
by means of diamide, as shown in Figure 6A. Therefore,
it is of importance to identify the chemical forms of
arsenic in RBCs.
Arsenic accumulates in control rat RBCs due to con-
taminating inorganic arsenic in food and the arsenic in
the hemolysate of control rats was detected mostly in the
void volume of a gel filtration column, as shown in Figure
7
A. The major sulfur peak (m/z 34) was also detected at
the void volume and GSH was detected as a small peak
at 13.3 min.
The DMA and DMAV peaks did not change on
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incubation with GSH (Figure 7B, parts a and b). How-
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ever, the DMA but not the DMA peak decreased on
incubation with a hemolysate, together with an increase
in the arsenic peak at the void volume (compare Figure
7
, part c in panel B with part b in panels B and A). GSH
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further enhanced the decrease in the DMA peak and
the increase in the arsenic peak at the void volume
(
compare Figure 7B, part d with part c). On the other
V
hand, DMA was not affected by incubation with GSH
and the hemolysate, as shown in Figure 7C. These results
suggest that DMA , but not DMA , forms a complex with
constituent(s) in RBCs in the presence of GSH, and that
arsenic is retained in RBCs in the form of a high
molecular weight complex.
F igu r e 6. Effect of glutathione oxidation with diamide on the
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uptake of dimethylarsinous acid (DMA ) by rat RBCs. Rat
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RBCs in Tris-HCl buffered saline (10%) were incubated with
different concentrations of diamide for 20 min at 37 °C, and then
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V
with DMA (A) and DMA (B) for 1 h at 37 °C. The distribution
of arsenic in the medium was determined on a cation exchange
column by the HPLC-ICP MS method. The vertical bar indicates
the detection level for arsenic.
Discu ssion
V
observable changes, as shown in Figure 6A. DMA was
not taken up by rat RBCs under the present conditions,
as shown in Figure 6B. These results suggest that DMA
The most significant difference in the metabolism of
arsenic among diverse animals has been recognized to
be that in the distribution of arsenic in RBCs of tolerant
rats and sensitive hamsters, arsenic being distributed
and retained mostly in rat RBCs (14, 15), but being
distributed slightly in RBCs and excreted into the urine
in the case of hamsters (16-18). However, the mecha-
nism underlying the difference in the metabolism has not
been elucidated. Therefore, the present study was focused
on the process of uptake of arsenic by RBCs based on
the speciation of arsenic at each metabolic step.
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is not retained in RBCs in the absence of GSH. However,
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the intensity of the DMA peak did not change with the
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presence of diamide, suggesting that DMA was not
oxidized even under GSH-depleted conditions in rat
RBCs.
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DMA incubated with hamster RBCs changed to
V
DMA , as shown in Figure 4B. This observation can be
explained by that DMAIII is oxidized to DMA in RBCs
V
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and then effluxed into the medium because DMA is
retained less effectively under less reductive conditions
The speciation of arsenic by HPLC-ICP MS, HPLC-
HG-AFS, or other methods has been carried out for
(Figure 6A). However, at the same time, the intensity of

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Difference in Arsenic (DMA ) Uptake by RBCs
Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1451
Sch em e 2. P r op osed Mech a n ism s Un d er lyin g th e
Up ta k e a n d Reten tion of Dim eth yla ted Ar sen ic
(
DMAs) by Red Blood Cells (RBCs)
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the retention of DMA by RBCs decreased with depletion
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of GSH, and DMA formed a complex with protein(s) in
RBCs, especially in the presence of GSH. These observa-
III
tions can be explained as follows: DMA forms a
complex with sulfur-rich specific protein(s) that can be
reduced by GSH, and then the complex is retained in
RBCs. However, when the concentration of GSH is not
F igu r e 7. Formation of a high molecular weight complex
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sufficient to reduce the specific protein(s), DMA is
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between arsenic of dimethylarsinous acid (DMA ) and protein-
V
oxidized to DMA through reduction of the specific
(s) in rat RBCs. Rat RBCs were lysed by freezing and thawing,
V
protein(s), DMA being effluxed, as proposed in Scheme
and the resulting lysate was centrifuged at 15000g for 20 min
to give a supernatant fraction (A). DMA (B) and DMAV (C)
were incubated in the supernatant fraction in the presence or
absence of glutathione (GSH) at 37 °C for 1 h. The distributions
of arsenic and sulfur (S) were determined on a gel filtration
column by the HPLC-ICP MS method at m/z 75 and 34,
respectively. The vertical bar indicates the detection level for
arsenic.
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2. Then, the animal species difference in the retention/
efflux of arsenic by RBCs can be attributed to the
difference in the specific protein(s) and/or GSH concen-
tration in RBCs.
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The uptake of DMA by RBCs in Figures 4 and 5 can
be divided into two distinct processes, i.e., the uptake of
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III
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DMA and the oxidation of DMA to DMA , followed
V
understanding the mechanism underlying the metabo-
by the efflux of DMA . The former process took place
lism of arsenic in the body and in the ecosystem (19-22,
faster in the order of rat > hamster > human, but the
latter process in the order of hamster > human > rat.
The animal species differences in these two processes
lead to variation in the metabolism and toxicity of arsenic
in addition to the differences in the reduction and
methylation processes in the liver. Therefore, it should
be noted that the uptake of DMA by RBCs is an
additional contributing factor to the reduction and me-
thylation capacity for the animal species difference in the
metabolism and toxicity of arsenic.
III
V
3
2, 33). The separation, into DMA and DMA , and
detection of DMA seem to shed new light on the mech-
anism underlying the toxicity of arsenic because of the
proposed metabolic pathway for arsenic in Scheme 1. In
III
fact, DMA has been demonstrated in the urine of people
inhabiting in an arsenic-contaminated area (19-22).
The present study revealed for the first time that
III
V
DMA , but not DMA , is the chemical form of arsenic
III
taken up by RBCs, and further that the uptake of DMA
by RBCs is dependent on the animal species. Uptake by
RBCs or excretion at the glomerulus seems to determine
the metabolic destination and distribution of small mo-
lecular weight DMA, and slower uptake by RBCs in vitro
suggests lower uptake by RBCs and higher excretion into
the urine in vivo. Further, the lower uptake by RBCs may
cause the higher incidence of the distribution of DMAIII
to other organs/tissues, which may explain the higher
incidence of the toxicity of arsenic in animals with less
arsenic distributed in RBCs. Although more data for
diverse animal species are required, the present observa-
Ack n ow led gm en t. This study was supported by
Grants-in-Aid of Ministry of Education, Science, Technol-
ogy, Sports and Culture (no. 12470509).
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TX015537K