Oral administration of diphenylarsinic acid, a degradation product of chemical warfare agents, induces oxidative and nitrosative stress in cerebellar Purkinje cells

Article abstract of DOI:10.1016/j.lfs.2007.09.030

A new clinical syndrome with prominent cerebellar symptoms in patients living in Kamisu City, Ibaraki Prefecture, Japan, is described. Since the patients ingested drinking water containing diphenylarsinic acid (DPA), a stable degradation product of both diphenylcyanoarsine and diphenylchloroarsine, which were developed for use as chemical weapons and cause severe vomiting and sneezing, DPA was suspected of being responsible for the clinical syndrome. The purpose of the present study was to elucidate prominent cerebellar symptoms due to DPA. The aim of the study was to determine if single (15 mg/kg) or continuous (5 mg/kg/day for 5 weeks) oral administration of DPA to ICR-strain mice induced oxidative and/or nitrosative stress in their brain. Significantly positive staining with malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) was observed in the cerebellar Purkinje cells by repeated administration (5 mg/kg/day) with DPA for 5 weeks that led to the cerebellar symptoms from a behavioral pharmacology standpoint and by single administration of DPA (15 mg/kg). Furthermore, it is possible that the production of 3-NT was not caused by peroxynitrite formation. The present results suggest the possibility that arsenic-associated novel active species may be a factor underlying the oxidative and nitrosative stress in Purkinje cells due to exposure to DPA, and that the damage may lead to the cerebellar symptoms.

Full text of DOI:10.1016/j.lfs.2007.09.030

Available online at www.sciencedirect.com
Life Sciences 81 (2007) 1518–1525
www.elsevier.com/locate/lifescie
Oral administration of diphenylarsinic acid, a degradation product
of chemical warfare agents, induces oxidative and
nitrosative stress in cerebellar Purkinje cells
a
a
b
c
d
d
Koichi Kato , Mutsumi Mizoi , Yan An , Masayuki Nakano , Hideki Wanibuchi , Ginji Endo ,
e
f
g
a,
⁎
Yoko Endo , Mikio Hoshino , Shoji Okada , Kenzo Yamanaka
a
Research Unit of Environmental Toxicology and Carcinogenesis, Nihon University College of Pharmacy, Chiba, Japan
Department of Radiation Effect, Institute of Radiation Medicine, Shandong Academy of Medical Sciences, Jinan, China
b
c
National Hospital Organization Chiba Medical Center, Chiba, Japan
d
Osaka City University Medical School, Osaka, Japan
e
Research Center for Occupational Poisoning, Tokyo Rosai Hospital, Tokyo, Japan
f
The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan
g
Professor Emeritus, University of Shizuoka, Shizuoka, Japan
Received 17 June 2007; accepted 19 September 2007
Abstract
A new clinical syndrome with prominent cerebellar symptoms in patients living in Kamisu City, Ibaraki Prefecture, Japan, is described. Since
the patients ingested drinking water containing diphenylarsinic acid (DPA), a stable degradation product of both diphenylcyanoarsine and
diphenylchloroarsine, which were developed for use as chemical weapons and cause severe vomiting and sneezing, DPA was suspected of being
responsible for the clinical syndrome. The purpose of the present study was to elucidate prominent cerebellar symptoms due to DPA. The aim of
the study was to determine if single (15 mg/kg) or continuous (5 mg/kg/day for 5 weeks) oral administration of DPA to ICR-strain mice induced
oxidative and/or nitrosative stress in their brain. Significantly positive staining with malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) was
observed in the cerebellar Purkinje cells by repeated administration (5 mg/kg/day) with DPA for 5 weeks that led to the cerebellar symptoms from
a behavioral pharmacology standpoint and by single administration of DPA (15 mg/kg). Furthermore, it is possible that the production of 3-NT
was not caused by peroxynitrite formation. The present results suggest the possibility that arsenic-associated novel active species may be a factor
underlying the oxidative and nitrosative stress in Purkinje cells due to exposure to DPA, and that the damage may lead to the cerebellar symptoms.
©
2007 Elsevier Inc. All rights reserved.
Keywords: Diphenylarsinic acid; 3-Nitrotyrosine; Purkinje cells; Cerebellar symptom; Oxidative stress; Nitrosative stress
Introduction
City, Ibaraki Prefecture, Japan, by diphenylarsenic compounds,
particularly diphenylarsinic acid (DPA), has been demonstrated
(Ishizaki et al., 2003). Furthermore, it is believed that DPA, the
main contaminant in the ground water, originated from
chemical warfare agents such as diphenylcyanoarsine and
diphenylchloroarsine; that is, it is a degradation product of
unstable diphenylcyanoarsine and diphenylchloroarsine. Ishii
et al. have reported that typical symptoms of DPA toxicity are
cerebellar dysfunction accompanied by hemokinetic dysfunc-
tion (oligohemia) as well as neurological impairment that
includes ataxia myoclonus and diplopia (Ishii et al., 2004).
However, the mechanisms of the dysfunction so far remain
Diphenylarsinous compounds such as diphenylcyanoarsine
and diphenylchloroarsine were developed during World War II
as chemical warfare agents with the aim of eliminating the will
of a soldier to fight (Pearson and Magee, 2002). A variety of
these chemical warfare agents were most likely abandoned by
the old Japanese Imperial Army in Japan after the end of World
War II. Recently, contamination of ground water in Kamisu
⁎
Corresponding author. Tel.: +81 47 465 6077; fax: +81 47 465 6077.
E-mail address: yamanaka@pha.nihon-u.ac.jp (K. Yamanaka).
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.09.030

K. Kato et al. / Life Sciences 81 (2007) 1518–1525
1519
unknown. A recent study reported that the toxicity by DPA was
exacerbated by sulfhydryl compounds (Ochi et al., 2004),
suggesting that a reduced form of DPA caused by sulfhydryl
compounds, trivalent diphenylated arsenic, but not DPA itself,
might be responsible for the toxic actions. This is also suggested
from the case of DPA; trivalent dimethylated arsenic, a
metabolite of dimethylarsinic acid, has greater toxicity than
pentavalent dimethylarsinic acid.
Although inorganic arsenics such as arsenite and arsenate are
known to cause carcinogenesis in skin, lung, and bladder, their
carcinogenic mechanisms remain obscure (IARC, 2004).
However, it was established by the International Agency for
Research on Cancer (IARC) that there is sufficient evidence for
carcinogenicity induced by dimethylarsinic acid, a main
metabolite of inorganic arsenics, in animals (IARC, 2004). To
clarify the mechanism of arsenic carcinogenesis, in recent
decades we and other research groups have focused our
attention on dimethylarsinic acid. Oral administration of
dimethylarsinic acid in mice and rats has been found to induce
tumor promotion in skin (Yamanaka et al., 2000, 2001a), lung
reaction procedure for DPA synthesis is shown schematically in
Fig. 1. First, phenylarsonic acid (Wako Pure Chemical
Industries, Ltd, Osaka, Japan) was dissolved in 6 M HCl and
then reduced with sulfur dioxide, which was generated from
sodium bisulfide and conc. H SO , in the presence of a trace
2
4
amount of potassium iodide. The phenyldichloroarsine pro-
duced here was extracted in chloroform and purified by
distillation (Bp760 252–5 °C). Next, DPA was prepared using
the phenyldichloroarsine synthesized here. Distilled aniline
(0.2 mol) dissolved in acetic acid (250 ml) was added to NaNO2
(0.22 mol) under ice-cold conditions, and the mixture allowed
to stand for 15 min. Phenyldichloroarsine (0.2 mol) dissolved in
acetic acid (450 ml) was added to the mixture. Powdered copper
(I) chloride (CuCl) was slowly added to the reaction mixture,
which was then allowed to stand overnight at room temperature.
After the addition of water, the pH of the mixture was made
alkaline by adding NaOH. After filtration, the pH of the reaction
mixture was adjusted to 7.0 by adding diluted HCl before
filtering again. The pH of the mixture was then adjusted to
approximately 4.0. The precipitates (DPA) produced were
collected and dissolved in chloroform. DPA was crystallized
from MeOH and water. The DPA synthesized was identified by
(
Yamanaka et al., 1996), and bladder (Wanibuchi et al., 1996),
leading to the conclusion that induction of the promotion action
is closely related to oxidative stress via the production of
reactive oxygen species (Yamanaka et al., 2001b; Mizoi et al.,
1
mass spectrometry, H-NMR, IR spectrometry, and LC-ICP-
MS (estimated purity N99%).
2
005). Therefore, it cannot be denied that organoarsinic acid
Diphenylarsinous iodide (DPI) was synthesized as follows:
DPA (0.1 mol) synthesized here was dissolved in 6 M HCl and
then reduced with sulfur dioxide, which was generated from
sodium bisulfide and conc. H SO , in the presence of potassium
toxicity may be related to the induction of oxidative stress.
In the present study, we have attempted to clarify the
mechanism of the cerebellar dysfunction caused by DPA, and
concluded that the induction of oxidative stress by DPA may
participate in the cerebellar dysfunction.
2
4
iodide (1.2 mol). The DPI produced was extracted in
chloroform and purified by distillation (Bp 173–5 °C). DPI
2
dissolved in small amounts of acetonitrile was used in in vitro
experiments (Western blot analysis) for production of superox-
ide anion and 3-NT as mentioned below.
Materials and methods
Caution: Since DPA and phenyldichloroarsine, a synthetic
intermediate of DPA, and diphenylarsinous iodide are highly
toxic, extreme care should be exercised when handling these
compounds.
DPA and other chemicals: DPA was synthesized by
modification of a method described by Takahashi (1952). The
Animals: All animal experiments were performed in
accordance with the Guidelines for Regulation of Animals
provided by the Experimentation Committee of Nihon Univer-
sity. Five-week-old male ICR-strain mice weighing approxi-
mately 25 g were obtained from Japan SLC (Hamamatsu,
Japan) through Sankyo Laboservice Corporation, Inc. (Tokyo,
Fig. 1. Schematic representation of the DPA synthesis procedure.

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K. Kato et al. / Life Sciences 81 (2007) 1518–1525
2
Japan). The mice were housed 3 per cage under SPF conditions
at 23 °C and 55% humidity with a 12-h light/dark-cycle. The
mice were given free access to food and drinking water.
Administration of DPA: DPA solutions (1.8 mg/ml DPA)
were prepared by dissolving DPA in 0.05 M NaOH and then
adjusting the pH to approximately 8.0 with 0.05 M HCl. The
mice were administered DPA at a single dose (2.5–15 mg/kg for
per cm of membrane for 30 min in 0.175 M tris, 1.344 M
glycine buffer containing 10% methanol. The membrane was
blocked with 5% dried defatted milk and 0.05% Tween-20 in
PBS. The membrane was then incubated with a 1:2000 dilution
of monoclonal antibody of 3-NT (Upstate, NY) in PBS
containing 0.05% Tween-20 overnight at 4 °C. Bound antibody
was detected using a biotin–streptavidin system with biotiny-
lated anti-mouse IgG.
2
4 h) or repeated dose (5 mg/kg/day for 5 weeks) by intragastric
gavage.
Thiobarbituric acid-reactive substances (TBA-RS): The cere-
Statistics: The level of significance between different groups
was calculated by analysis of variance (ANOVA) and Fisher's
PLSD-test, as described in our previous paper (Yamanaka et al.,
2001a). The PC packaged software used in the statistical
analysis was Origin® Pro, version 7.5J (OriginLab Corporation,
Northampton, MA, USA).
brum and cerebellum were excised at 24 h after exposure of the
mice to DPA (2.5–15 mg/kg) and homogenized (30% w/v) in
1
3
.15% KCl. Each homogenate was mixed with acetate buffer (pH
.5) and 0.8% TBA solution. The samples were placed in boiling
water for 60 min and then centrifuged at 1500 rpm (2,000 ×g) for
5 min after being mixed with n-butanol and vortexed. The levels
1
Results and discussion
of TBA-RS were quantified by spectrophotometry using a molar
extinction coefficient for the TBA-derivative of MDA
In both male and female rats, the no-observed effect level
(NOEL) of DPA is estimated to be 0.3 and 0.8 mg/kg/day,
respectively [Toxicological reports of DPA by Ministry of the
Environment of Japan, URL: http://www.env.go.jp/en/]. This
value is estimated to be 10 times higher than that in humans.
Moreover, in vivo studies performed by a research group led by
Professor Fushiki at Kyoto Prefectural University of Medicine
have confirmed that repeated oral administration (5 mg/kg/day)
for 5 weeks of DPA induces cerebellar dysfunction in male ICR-
strain mice as well as in rats (personal communication).
To elucidate the toxic mechanisms of a new clinical syn-
drome exhibiting prominent cerebellar symptoms due to
exposure to DPA, we first used a subchronic dose (5 mg/kg/
day) of DPA for 5 weeks and a single acute dose (15 mg/kg) of
DPA. Fig. 2 presents a light micrograph of cerebellum stained
with hematoxylin and eosin. The only instance of morpholog-
ical changes, including cell-nuclear atrophy (pyknosis), was
observed in the cerebellum, particularly in cerebellar Purkinje
cells (arrows), of mice administered DPA (Fig. 2b, c) but not in
control mice (Fig. 2a). The pyknosis of cerebellar Purkinje cells
was also observed at 5 weeks after the start of the repeated oral
administration of DPA (Fig. 2c) as well as at 24 h after acute
single administration (15 mg/kg) (Fig. 2b). Even the maximum
dose of 15 mg/kg did not kill any mice at 24 h after the
administration and histopathological changes were not observed
in any organs and tissues, including the cerebrum (data not
shown). Accordingly, the prominent cerebellar symptoms due
to DPA may be closely related to damage to cerebellar Purkinje
cells. Furthermore, we believe that it may be possible to reveal
the mechanism of the prominent cerebellar symptoms using a
biochemical approach using mice administered a single acute
dose of DPA (15 mg/kg).
5
−1
−1
(
1.56×10 M cm at 532 nm) (Ohkawa et al., 1979).
Immunohistochemical detection of malondialdehyde
MDA)-adducts and 3-NT in cerebellar sections: Immunohis-
(
tochemical studies on MDA-protein adducts and 3-NT in
cerebellar sections were performed on 3 μm formalin-fixed and
paraffin-embedded serial tissue sections mounted on 2% 3-
aminopropyltriethoxysilane-coated slides. The subsequent im-
munohistochemical detection was based on the method
described in our previous report (An et al., 2004) using MDA
(
NOF Corporation, Tokyo, Japan) and 3-NT (Upstate, NY,
USA) monoclonal antibody.
Determination of superoxide dismutase (SOD) activity: A
commercial kit (SOD Assay Kit — WST, Dojindo Laboratories,
Kumamoto, Japan) was used to measure SOD in the tissue after
DPA administration. Cerebral and cerebellar homogenates were
centrifuged at 12,000 rpm (9000 ×g) at 4°C for 30 min. The
supernatants were assayed for SOD activity, which was
measured by the inhibition of auto-oxidation of 2-(4-iodophe-
nyl)-3-(4-nitropheny)-5-(2,4-disulfo- phenyl)-2H-tetrazolium,
monosodium salt (WST-1) by spectrophotometry at 450 nm.
Determination of glutathione peroxidase (GSH-Px) activity:
GSH-Px activity was measured by the conversion of NADPH to
+
NADP in the presence of H O (Paglia and Valentine, 1967).
2
2
Cerebral and cerebellar homogenates were centrifuged at
2,000 rpm (9000 ×g) at 4 °C for 30 min. The supernatants
10 μl) were mixed with 0.98 ml of 100 mM sodium phosphate
1
(
buffer (pH 7.0) containing 2 mM GSH, 1 unit of GSH reductase,
and 0.2 mM NADPH. The reaction was started by adding 10 μl of
H O . GSH-Px activity was quantified by spectrophotometry using
2
2
6
−1
−1
a molar extinction coefficient for NADPH (6.22×10 M cm at
340 nm).
Electrophoresis and Western blot analysis: Bovine serum
albumin (BSA) (50 mg/ml) in 0.1 M phosphate buffer (pH 6.8)
was treated with 10 μM DPI, 100 μM NO and 10 μM DPI plus
00 μM NO, respectively. 3-NT-BSA standard was produced by
the addition of 5 mM peroxynitrite to BSA in 0.1 or 0.5 M
phosphate buffer (pH 6.8). The treated BSA solution was
applied to 8% SDS-PAGE and electroblotted onto a PVDF
Since our investigations on the mechanisms of toxicity and
carcinogenicity by arsenics have indicated that the induction of
oxidative stress plays an important role in these processes
(Yamanaka et al., 1991, 2001b; Mizoi et al., 2005), in the
present study, we focused our attention on the induction of
oxidative stress by oral administration of DPA. The values of
thiobarbituric acid-reactive substances (TBA-RS) were mea-
sured in the brain of mice after oral administration of DPA to
1
(
Hybond-P) membrane by a semidry transfer apparatus at 2 mA

K. Kato et al. / Life Sciences 81 (2007) 1518–1525
1521
components of the cerebellar cortex such as parallel fibers
themselves, glia cells and interneurons contain NO synthase
(nNOS) (Schmidt et al., 1992; Vincent and Kimura, 1992;
Southam et al., 1992). In particular, NO acts as a neurotrans-
mitter from the parallel fibers to the Purkinje cells (Daniel et al.,
1998) and is present in a sufficiently large amount around the
Purkinje cells. Accordingly, we considered the possibility that
new-type active species generated by the reaction of dipheny-
lated arsenic with NO caused the increase in TBA-RS values in
the cerebellum. These results also suggest that the induction of
oxidative stress might play an important role in prominent
cerebellar symptoms due to exposure to DPA.
To further elucidate the mechanism of the diphenylarsenic-
induced cerebellar oxidative damage, we performed a histo-
pathological examination. Fig. 4 shows micrographs of
cerebellar Purkinje cells immunostained with monoclonal
antibodies for 3-NT (a, b) and MDA (c, d), typical biomarkers
of nitrosative and oxidative stress, respectively. Positive
staining for 3-NT and MDA by these antibodies was confirmed
in cerebellar Purkinje cells of mice treated with DPA (at dose of
5
mg/kg/day) for 5 weeks (b, d) but not in the control mice (a, c).
These phenomena are observed even in the case of a single oral
dose of DPA (15 mg/kg) (data not shown). Furthermore,
quantitative evaluation of the immunohistochemical stains was
performed (Table 1). An apparent increase in positive-stained
cells for both 3-NT and MDA monoclonal antibodies was
observed.
One of the causes of the induction of oxidative stress may be
that the reactive oxygen species (ROS) produced are involved in
the oxidative stress. Therefore, we next attempted to clarify the
involvement of ROS in DPA-induced brain damage. Briefly, the
activities of the 2 ROS-eliminating enzymes, GSH-Px and
SOD, were measured. As shown in Table 2, oral administration
of DPA in mice resulted in an apparent increase in GSH-Px
activity in cerebellum compared to the control mice and the
activity was hardly suppressed even in the case of the addition
of sodium azide, an inhibitor of catalase (data not shown),
suggesting a biochemical event accompanying the increase in
Fig. 2. Hematoxylin–eosin staining of cerebellum in mice after DPA
administration. (a) control, (b) 24 h after single administration of DPA
(
15 mg/kg), (c) repeated administration of DPA (5 mg/kg/day) for 5 weeks. The
arrows indicate pyknotic Purkinje cells. The bars represent 100 μm.
determine if diphenylated arsenics, similarly to dimethylated
arsenics, also induce oxidative stress. Fig. 3 shows the TBA-RS
values in the cerebellum and cerebrum of mice at 24 h after oral
administration of DPA (2.5–15 mg/kg). TBA-RS values in
cerebellum increased significantly in a dose-dependent manner,
whereas those in cerebrum did not increase. Furthermore, the
increase in the TBA-RS values was not observed in any other
tissues. The fact that the brain is a target for the lipid
peroxidation by diphenylated arsenic makes the accumulation
of diphenylated arsenics in brain understandable because it is
reasonable to assume that DPA should also have greater lipo-
philicity and is then reduced to trivalent diphenylated arsenic
with SH compounds in the brain. On the other hand,
Fig. 3. TBA-RS in cerebellum of mice 24 h after DPA administration. Male ICR-
strain mice were administered DPA (2.5–15 mg/kg). Each bar represents the
⁎
mean±SE (n=6). Significant difference (pb0.01) from control.

1522
K. Kato et al. / Life Sciences 81 (2007) 1518–1525
Fig. 4. Distribution of modified protein-adducts in cerebellum of mice by repeated oral administration of DPA (5 mg/kg/day) for 5 weeks. (a) and (b); 3-NT monoclonal
antibody-treated cerebellar tissues. (c) and (d); MAD-adduct monoclonal antibody-treated cerebellar tissues. (a) and (c) indicate tissues from control mice. (b) and (d)
indicate tissues from mice administered DPA. The arrows in (a), (b), and (d) indicate positively-stained Purkinje cells. The bars represent 100 μm.
TBA-RS values (the formation of lipid peroxidation but not
hydrogen peroxide) shown in Fig. 3. However, SOD activity
was not increased significantly in cerebellum compared to the
control mice.
Therefore, we attempted to perform an in vitro experiment using
DPI, a model compound of trivalent diphenylated arsenic. We
predicted that DPI may be hydrolyzed to diphenylarsinous acid
as expressed in formula (1) and then confirmed by HPLC-ICP
MS analysis that DPI is finally oxidized to DPA (data not
shown).
Here, a discrepancy has arisen between the formation of 3-
−
NT and slight increase in superoxide anion (O ), because 3-NT
2
is generally thought to be formed via the production of
−
−
ðC6H5Þ AsI þ H2O→ðC6H6Þ AsOH þ HI
ð1Þ
2
2
peroxynitrite (ONOO ) by reaction of NO with O (Kobzik and
2
Schmidt, 1996). A recent study has indicated that highly toxic
chemicals generated by the reaction of DPA with sulfhydryl
compounds, e.g., GSH, could have cytotoxic and genotoxic
actions (Ochi et al., 2004). It seems that the substances that
induced these actions may be produced by the reduction of DPA
to trivalent diphenylated arsenic, although their identities are
still unclear.
Based on the findings obtained from our in vitro experiment
using dimethylarsinous iodide (Mizoi et al., 2005), it may be
easy to explain the hydrolyzing and then the oxidizing from DPI
to DPA as well. The results of an in vitro study using the
cytochrome c method (Yamanaka et al., 1990) indicated that the
presence of trivalent diphenylated arsenic under aerated
−
conditions could not produce O (data not shown), suggesting
2
Generally, it is believed that O 2− is produced by a one-
electron reaction of O2 with trivalent diphenylated arsenic
produced from the reaction of DPAwith sulfhydryl compounds.
Table 2
The activities of ROS-eliminating enzymes in cerebellum of mice after the
administration of DPA
Table 1
Control
DPA
Formation of 3-NT-adducts in cerebellar Purkinje cells after oral administration
at dose of 5 mg/kg/day for 5 weeks
a
GSH-Px
SOD
6.2±1.1
7.9±0.6
10.4±0.6
8.8±0.2
Positive-stained cells/Purkinje cells (%)
Male ICR-strain mice were treated with DPA (15 mg/kg, p.o.). GSH-Px and
SOD activity in cerebellum of mice 24 h after the administration were expressed
as ×10 units/mg protein and inhibition(%)/mg protein, respectively. Data
3-NT
MDA
−
3
Control
DPA
125/161 (77.6)
172/175 (98.3)
61/161 (37.9)
155/175 (88.6)
represent the mean ±SE (n=4–5).
a
Significant difference (pb0.01) from control levels.

K. Kato et al. / Life Sciences 81 (2007) 1518–1525
1523
Fig. 5. Determination of nitrating tyrosine residues in BSA reacted with DPI and nitric oxide. BSA (50 mg/ml) was treated with DPI, NO, and DPI plus NO in 0.1 M
phosphate buffer (pH 6.8), respectively. Positive control (BSA including 3-NT residues) was produced by the treatment of peroxynitrite to BSA in 0.1 or 0.5 M
phosphate buffer (pH 6.8). These reaction mixtures were applied to 8% SDS-PAGE. 3-Nitrotyrosine residues in BSAwere detected by Western blot analysis using anti-
3-NT monoclonal antibody.
−
that one-electron reduction from O to O with diphenylarsi-
nous acid is fairly slow.
This result could suggest possible production mechanisms of
3-NT by a new active nitrosative species produced from the
reaction between trivalent diphenylated arsenic and NO in the
presence of molecular oxygen but not by peroxynitrite. Their
possible production mechanisms are summarized in Fig. 6.
Diphenylarsinous acid reacts with molecular oxygen in the
presence of NO, and could produce the arsenic-associated active
species (I). This species is a derivative of peroxynitrite
combined with trivalent diphenylated arsenic and reacts with
tyrosine to ultimately form 3-NT. Since except for peroxynitrite
the only species capable of forming 3-NT reported so far is
2
2
Accordingly, the formation of 3-NT in Purkinje cells may be
due to a novel production mechanism that is independent of
ONOO . Therefore, we postulated the formation of a new type
−
of NO donor by the reaction of trivalent diphenylated arsenic
2
and NO. DPI and NO were allowed to react with BSA in the
phosphate buffer for 1 h. After electrophoresis and Western blot
analysis, the reaction mixture was immunostained with
nitrotyrosine monoclonal antibody (Fig. 5). The BSA interacted
with DPI and NO was positively stained by 3-NT monoclonal
antibody in a comparison with the positive control (treated with
peroxynitrite).
NO Cl (Eiserich et al., 1998), the new-type active species (I)
2
shown here is worth noting. Furthermore, the induction of
Fig. 6. Possible production mechanisms of 3-NT produced by reaction of tyrosine with diphenylarsinous acid and NO in the presence of molecular oxygen.

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species (I).
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intercellular molecular messenger involved in neuromodulation
and blood circulation (Moncada and Higgs, 1991). Many
studies examining cerebellar function have indicated that the
diffusion of NO from parallel fiber synapses onto Purkinje cells
is necessary for explaining motor learning (Daniel et al., 1998;
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cerebellum generally show the highest levels of nNOS
immunoreactivity and mRNA (Bredt et al., 1990, 1991).
There is a particularly large amount of nNOS immunoreactivity
in the cerebellar cortex of mice (Gotti et al., 2005). The NO
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dysfunction accompanying oligohemia might arise due to a
decrease in the NO. In fact, the present data suggest that
cerebellar dysfunction due to exposure to diphenylated arsenic
might be based on the damage of Purkinje cells by the induction
of oxidative and nitrosative stress derived from the arsenic-
associated active species (I) (Fig. 6) and also on a decrease in
NO levels in Purkinje cells following the production of this
active species (I).
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This work was supported in part by Grant-in-Aid for
Scientific Research (A) from Japan Society for the Promotion
Science (16209021) from the Ministry of Education, Culture,
Sports, Science and Technology and “Academic Frontier”
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