ANALYTICAL SCIENCES OCTOBER 2009, VOL. 25
1189
2009 © The Japan Society for Analytical Chemistry
Reviews
Toxicometallomics for Research on the Toxicology of Exotic
Metalloids Based on Speciation Studies
Yasumitsu OGRA
Laboratory of Chemical Toxicology and Environmental Health, Showa Pharmaceutical University,
3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194–8543, Japan
Tellurium and antimony are widely used in industry because of their unique chemical and physical properties. Although
these metalloids, which belong to period 5 of the periodic table of elements, are known to be non-essential and harmful,
or the so-called “exotic” elements, little is known about their toxic effects and metabolism. The present review describes
the role of speciation in considering the metabolism of tellurium and antimony from the viewpoint of toxicometallomics.
Inorganic tellurium in the form of tellurite is reduced and simply methylated in the body. Rat red blood cells accumulate
tellurium in the form of dimethylated tellurium, and tellurium is excreted into urine as trimethyltelluronium. Although
selenium, which belongs to the same group as tellurium, is known to be excreted in the form of selenosugar as the major
urinary metabolite, tellurosugar was not detected by an inductively coupled plasma–mass spectrometer hyphenated with
an HPLC. Speciation studies revealed that the major metabolic pathway of antimony is oxidation in human and rat, and
methylation also occurs as a minor metabolic pathway in humans.
(Received August 21, 2009; Accepted September 7, 2009; Published October 10, 2009)
1 Introduction
2 Speciation in Relation to Metallomics
2·1 Metallomics and toxicometallomics
2·2 Speciation and hyphenated techniques
3 Metabolism of Tellurium
3·1 Tellurium in biology, toxicology, and
medicine
3·2 Metabolism of tellurium based on the
characterization of tellurium species by
speciation studies
1189
1190
1191
1 Introduction
Metalloids possess properties that are intermediate between
those of metals and nonmetals. They are widely used in industry
because of their unique chemical and physical properties.
Recently, tellurium (Te) and antimony (Sb) have been used as
an alloy in industrial materials, such as phase-change optical
magnetic disks, digital versatile disk-random access memory
(DVD-RAM), and DVD-recordable disks (DVD-RW).1
Although these metalloids, which belong to period 5 of the
periodic table of elements, are known to be non-essential and
harmful, or the so-called “exotic” elements, little is known about
their toxic effects and metabolism. In addition, since these
metalloids exhibit unique pharmacological effects, they are also
used in medicine. The diverse toxicological and pharmacological
effects of these metalloids depend on their chemical species in a
biological system. Furthermore, metalloids form organometallic
E-mail: [email protected]
4 Metabolism of Antimony
4·1 Antimony in biology, toxicology, and
medicine
4·2 Metabolism of antimony based on the
characterization of the antimony species by
speciation studies
5 Conclusions and Perspectives
6 Acknowledgements
7 References
1192
1194
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1194
compounds having carbon-metalloid covalent bond(s) in the
metabolic pathways of animals. Therefore, it is necessary to
identify the organometallic compounds to clarify the metabolic
pathways of the metalloids and to understand their toxicological
or pharmacological effects.2,3 Speciation is one of the most
commonly employed analytical techniques for the separation
and detection of metal/metalloid-containing species in biological
samples.4–7 A hyphenated technique that combines HPLC with
an inductively coupled plasma–mass spectrometer (ICP-MS) is a
powerful tool for the speciation of metal/metalloid-containing
biomolecules.8,9
The present review describes the role of speciation in
considering the metabolism of Te and Sb from the viewpoint
of toxicometallomics.
The mechanisms underlying the
discriminatory metabolism of Te and Sb from selenium (Se) and
arsenic, which are, respectively, metalloids of the same group,
but appearing in period 4 of the periodic table, are also
discussed.
1190
2 Speciation in Relation to Metallomics
2·1 Metallomics and toxicometallomics
The newly coined term “metallome” is defined as “the entirety
of metal and metalloid species present in a cell or tissue type,
their identity, quantity and localization.”10,11 Thus, metallomes
consist of metals/metalloids in diverse chemical forms in a
biological system. Meanwhile, the term “ionome” was coined
in the field of systems biology.12 Contrary to metallomes,
ionomes consist of free metals (ion forms) in a cell or tissue
type. Hence, qualification (identification of metal/metalloid
species) and quantification (determination of metal/metalloid
content) are simultaneously required in metallome analysis.
The research field dealing with the study of metallomes and
their relationships with genomes and proteomes is referred to as
metallomics.13
Metallomics is very much connected to
multidisciplinary research fields, including analytical chemistry,
nuclear
chemistry,
geochemistry,
biology,
nutrition,
pharmacology, toxicology, and medicine.
In particular,
toxicology is intricately related to metallomics because of the
huge number of people who have suffered from metal poisoning
in the past, and many who suffer from the same at present.
Methylmercury and cadmium are the causative substances of
Minamata disease and Itai-itai disease, respectively, both of
which are pollution-related diseases that have occurred in Japan.
Around 60 million people suffer from arsenic toxicity due to the
ingestion of water naturally contaminated with inorganic arsenic
species in the world, mainly in Asian countries.14 Although it is
known that non-essential and excess amounts of essential
metals/metalloids have toxic effects, some metals/metalloids
having unique chemical and physical properties are currently
utilized in industry without sufficient and necessary risk
assessment. Indeed, the toxicological effects of newly used
metalloids, such as Te and Sb, are less understood than those of
Se and arsenic, which are metalloids of the same group as Te
and Sb, respectively, appearing in period 4 of the periodic table.
Toxicometallomics is a coined word concerning toxicology and
metallomics, and is considered to be a part of metallomics for
metallomes comprising toxic metal/metalloid species in a
system. Thus, toxicometallomics studies of these unique and
non-essential “exotic metalloids” may contribute to minimizing
damage to human health and the ecosystem.
2·2 Speciation and hyphenated techniques
The term “speciation” of an element, including a metal or a
metalloid, is defined as the distribution of an element among
defined chemical species in a system, while the technique
“speciation” is defined as analysis to identify and/or measure
the quantities of one or more individual chemical species in a
sample.15 Since metallomes consist of metals/metalloids in
diverse chemical forms in a biological system, it is required that
metal/metalloid species should be separable by various
separation techniques and detectable with a specific detector for
identification and quantification. To perform the speciation of
metals/metalloids, hyphenated techniques are used.9
As mentioned above, hyphenated techniques for speciation
have to consist of two independent analytical techniques, i.e., a
separation technique based on the physical and chemical
properties of biomolecules containing metals/metalloids,
and a specific detection technique for the metals/metalloids.
High-performance liquid chromatography (HPLC) is one of the
most popular separation techniques used with hyphenated
techniques. HPLC has several advantages over other separation
techniques, such as gas chromatography (GC), gel electrophoresis,
ANALYTICAL SCIENCES OCTOBER 2009, VOL. 25
and capillary electrophoresis: 1) diverse techniques, such as gel
filtration, ion exchange, affinity, and reversed phase, are
available, and 2) the separation conditions are similar to
physiological conditions, particularly on a size-exclusion/gel
filtration column. On the other hand, inductively coupled
plasma–mass spectrometry (ICP-MS) has enabled multi-elemental
detection with extremely high sensitivity and isotope
discrimination, and is robust to diverse matrices in biological
samples.16 In addition, the eluate of HPLC can be directly
introduced into ICP-MS because the flow rate of regular bore
HPLC is proportional to that for introduction to ICP-MS.
Therefore, HPLC coupled with ICP-MS (HPLC-ICP-MS) is the
most widely used technique for speciation.
However, there remains a critical disadvantage in the
identification of unknown metalloid-containing metabolites by
HPLC-ICP-MS. Although ICP-MS is sensitive to target
elements, and is robust to matrices, it does not provide any
molecular information of metalloid-containing metabolites.
Thus, the identification of unknown metabolites by
HPLC-ICP-MS is limited to situations where authentic metalloid
species are available. Even if the standard compound is
available, the identification by HPLC-ICP-MS is still an indirect
method; namely, it is based on a comparison of the retention
times of a standard compound and an unknown metabolite.
Thus, it is mandatory to use at least two different chromatographic
mechanisms for the unambiguous identification of unknown
metabolites by HPLC-ICP-MS.
As an alternative to
HPLC-ICP-MS, electrospray ionization (ESI)–tandem mass
spectrometry (MS-MS) is used to identify unknown
metal/metalloid containing metabolites.17–19 ESI is a softer
technique than ICP, and can provide molecular information.
In addition, MS-MS enables structure elucidation. However,
ESI-MS-MS has a number of weak points compared with
ICP-MS. First, the detection limit of ESI-MS-MS is generally
inferior to that of ICP-MS. Second, ESI is severely affected by
the sample matrix. To counter this problem, ESI-MS-MS is
also coupled with HPLC. Therefore, the complementary use of
HPLC-ICP-MS and HPLC-ESI-MS-MS is a powerful tool for
the speciation and identification of metalloid-containing
metabolites.2,6
GC is another technique for the speciation of metalloids.20
GC-MS and GC coupled with an element-specific detector, such
as ICP-MS, have been applied to the detection of volatile Te
and Sb species in biological samples. By introducing a hydride
generation (HG) step prior to GC, non-volatile inorganic Te and
Sb species, such as tellurite, tellurate, antimonite, and antimonate,
can be also applied to GC.
As mentioned above, a highly sensitive detection of metalloids
and an identification of metalloid containing species by mass
spectrometry, i.e., ICP-MS and ESI-MS-MS have paved a road
to toxicometallomics for exotic metalloids. Previous studies
addressing the speciation of Te and Sb species in biological
samples are summarized in Table 1. Due to the anionic behavior
of inorganic Sb, such as antimonite (Sb(III)) and antimonate
(Sb(V)), anion-exchange chromatography is the main
methodology for speciation. However, good chromatographic
separation and recoveries have been reported from reversed-phase
(Hypersil C18) and gel filtration (HEME-BIO) columns.21 The
gel filtration column is able to separate antimony(V) citrate
from free Sb(V) ions in urine.22 Other chromatographic
mechanisms, such as multi-mode gel filtration and
cation-exchange chromatography, are also effective to separate
organic Te metabolites.23 As mentioned above, the advantage
of utilizing a gel filtration column is that the separation
conditions are similar to the physiological conditions. In
ANALYTICAL SCIENCES OCTOBER 2009, VOL. 25
Table 1
1191
Te and Sb species in biological samples detected with hyphenated techniques
Sample
Rat urine
Species
(CH3)3Te+ and
synthetic standard
Rat blood serum
Dimethylated Te
compounds
Human urine, milk powder Te(IV) and Te(VI)?
Urine from people
exposed occupationally
and standard reference
material
Urine and plasma of
patients treated with
meglumine antimoniate
Sb(V)-spiked urine
Sb(III), Sb(V), and
(CH3)3SbCl2
Meglumine
antimoniate, Sb(III),
Sb(V), and
unknown
Sb(III), Sb(V), and
antimony(V) citrate
HPLC columna
Mobile phase
(a) Shodex GS-320HQ (300 ×
7.5), multi-mode gel filtration
(b) Shodex NN-614 (150 × 6.0),
cation exchange
Shodex GS-520HQ (300 × 7.5),
multi-mode gel filtration
Hamilton PRP-X100 (250 ×
4.1), anion exchange
(a) Hamilton PRP-X100
(250 × 6.4), anion exchange
(b) Dionex IonPac AS14 (250 ×
4.0), anion exchange
(c) Cetac ION-120 (120 × 4.6),
anion exchange
Hamilton PRP-X100 (150 ×
4.1), anion exchange
(a) Hamilton PRP-X100
(250 × 4.1)
(b) BDS Hypersil C18 (150 ×
2.1), reversed phase
(c) HEMA-BIO (300 × 8.0), gel
filtration
Detector
(a) 50 mM ammonium
acetate, pH 6.5
(b) 50 mM ammonium
acetate, pH 8.0
50 mM Tris–HCl, pH 7.4
ICP-MS
ICP-MS
44
Gradient of 0.5 mM
ammonium citrate in 2%
methanol, pH 3.7, and
20 mM ammonium citrate
in 2% methanol, pH 8.0
(a) 20 mM EDTA, pH 4.7
ICP-MS
56
ICP-MS,
HG-ICP-MS
ICP-MS,
HG-ICP-MS
ICP-MS,
HG-ICP-MS
53
ICP-MS
57
ICP-MS,
ESI-MS-MS
ICP-MS,
ESI-MS-MS
ICP-MS,
ESI-MS-MS
21
(b) 1.25 mM EDTA, pH 4.7
(c) 2 mM ammonium
bicarbonate + 1 mM
tartaric acid, pH 8.5
20 mM EDTA, pH 4.7
(a) 20 mM EDTA + 2 mM
phthalic acid, pH 4.5
(b) 1 mM ammonium
formate + 10% methanol
(c) 10 mM ammonium
acetate + 10% methanol
addition, the column does not require the addition of chelators,
such as EDTA, tartaric acid, and phthalic acid, to achieve good
separation of Te and Sb species in biological matrices. Thus,
the use of a gel filtration column is the most preferable to
separate Te and Sb metabolites in their native forms.
3 Metabolism of Tellurium
Fig. 1
23
ICP-MS
a. Numbers in parentheses express column size in mm.
3·1 Tellurium in biology, toxicology, and medicine
Te is non-essential and hazardous, and its biological and
toxicological effects are little understood. Se, which belongs to
the same group as Te, is an essential element that forms an
active center in the formation of a selenol group on
selenocysteinyl residues in Se-requiring enzymes, i.e.,
selenoproteins.24,25 Thus, Te is expected to exist as Te-substituted
amino acids in biota. Some studies have reported Te metabolism
in bacteria, yeasts, and fungi; for example, tellurocysteine and
telluromethionine are found in proteins from such
microorganisms.
However, no telluroproteins have been
identified in animal cells to date.26–29 Consequently, Te is
considered to be a non-essential element in animals.
Toxicological information of Te is also limited. To draw
attention to Te toxicity, animal experiments were conducted
using extremely high doses of Te.30 The experiments showed
Te toxicosis in the animals, and the animals gave off a garlic-like
odor that originated from dimethyltelluride (DMTe) formed in
the body (Fig. 1).30 This pathway is quite similar to that of Se.
However, Se is mainly excreted into urine under physiological
conditions as selenosugar.31,32 Although Te is a non-essential
metalloid, it is expected to be metabolized in the same pathway
as that of an excess amount of Se, an essential metalloid. Thus,
Ref.
Structures of Te-containing metabolites and medicine.
1192
ANALYTICAL SCIENCES OCTOBER 2009, VOL. 25
one exciting task is to identify the urinary metabolites of Te,
because this may reveal the metabolic pathway of Te in animals.
As mentioned later, speciation has contributed to depicting the
metabolic chart of Te and highlighting the mechanisms to
discriminate the metabolic pathways of Te and Se in animals.
The organotellurium compound, trichloro(dioxoethylene-O,O′)tellurate (AS101), has diverse pharmacological effects both
in vivo and in vitro.33 AS101 has antitumor activity and is now
under clinical trial.
This compound also acts as an
immunomodulator, i.e., it inhibits the activation of interleukin-1β.
Interleukin-1β is activated by caspase-1, a member of the
cysteine protease family. The Te compound can selectively
bind to a sulfhydryl group in the cysteinyl residue located in the
active center of caspase-1. Thus, the inhibition of interleukin-1β
by the Te compound is explained as the interaction between the
compound and the activating enzyme.
3·2 Metabolism of tellurium based on the characterization
of tellurium species by speciation studies
As mentioned above, Te is expected to be metabolized in the
same pathway as that of an excess amount of Se, an essential
metalloid. Thus, the comprehension of Se metabolism may be
useful to consider Te metabolism. The Se metabolism currently
proposed is as follows: Diverse inorganic and organic Se species
are utilized as a nutritional source of Se, including selenite,
selenate and selenoamino acids.3 These species are converted
into a common metabolic intermediate, selenide, and selenide is
introduced into several pathways for the synthesis of
selenoproteins and the excretion of Se (Fig. 2). Details of the
mechanisms underlying the conversion of inorganic and organic
selenium compounds into selenide are available in reviews.3,6,34
Selenide has two excretion pathways, depending on the amount
ingested. Within the nutritional and low toxicity range of Se
ingested, selenide is primarily metabolized into selenosugar.32
Three species of selenosugars were detected as urinary Se
metabolites,
i.e.,
Se-methylseleno-N-acetyl-galactosamine
(MeSeGalNAc),
Se-methylseleno-N-acetyl-glucosamine
(MeSeGlcNAc),
and
Se-methylselenogalactosamine
(MeSeGalNH2).35,36
MeSeGalNAc is the most abundant
chemical form among them, and the other two species exist in
trace amounts.37,38 Indeed, only one Se peak corresponding to
MeSeGalNAc was detected in an HPLC-ICP-MS profile when a
urine sampled from a healthy subject was injected.
A second urinary Se metabolite appears when Se is ingested at
toxic levels. The metabolite was identified as trimethylselenonium
(TMSe) in literature.38–40 TMSe is simply the methylated form
of selenide, and thus its metabolic intermediates, such as
dimethylselenide (DMSe) and monomethylselenol (MMSe) or
dimethyldiselenide (DMDSe), which is the oxidized form of
MMSe, are also detected in urine and the breath because these
intermediates are volatile.41–43
Recently, the major urinary Te metabolite in rat, an
experimental animal, was identified by HPLC-ICP-MS and
ESI-MS-MS after tellurite, an inorganic Te compound, was
ingested.23 The Te metabolite was trimethyltelluronium (TMTe).
However, no peaks corresponding to Te-containing sugars, i.e.,
tellurosugars, were detected on HPLC-ICP-MS equipped with a
gel filtration column or a cation-exchange column. These
results based on speciation suggest that Te is discretely
metabolized from Se; namely, although the enzyme(s) catalyzing
the methylation of metalloids cannot distinguish Te from Se, the
enzyme(s) catalyzing the transfer of the sugar moiety to Se can
discriminate Te from Se (Fig. 2). At present, there is no
reasonable explanation as to why Se is excreted in two forms,
i.e., selenosugar and TMSe, while TMTe is the only urinary
Fig. 2
Se.
Proposed metabolic pathway of Te compared with that of
metabolite of Te. In addition to speciation studies, molecular
biological studies may also be needed to reveal the molecular
mechanisms of the discrimination in future studies.
The other characteristic of Te metabolism is the accumulation
of Te in red blood cells (RBCs) of rat. It is well known that
arsenic is accumulated markedly in rat RBCs.44 Meanwhile, it
was reported that Te is slightly accumulated in RBCs.45 Selenite
is incorporated into RBCs for reduction to selenide with
glutathione, which abundantly exists in RBCs. However, since
selenide is promptly effluxed into bloodstream and is bound to
albumin, Se is not accumulated in RBCs. Speciation studies by
HPLC-ICP-MS and ESI-MS-MS revealed that the chemical
form of accumulated Te is the dimethylated Te species, although
the precise chemical form has not yet been identified. In the
case of arsenic, the chemical form that is accumulated in RBCs,
is dimethylated arsenical (DMAs).44,46 Dimethylated Te is
specifically bound to hemoglobin-like DMAs. Thus, these
results suggest that the mechanism underlying Te accumulation
in RBCs may be similar to that underlying arsenic accumulation.
Because tellurite is not methylated in RBCs in vitro, dimethylated
Te is not formed in RBCs. The final methylation product of
arsenic is DMAs, whereas that of Te is TMTe, suggesting that
the metabolic intermediate in the Te methylation may be formed
in organs and is accumulated in RBCs.
4 Metabolism of Antimony
4·1 Antimony in biology, toxicology, and medicine
Although Sb is a non-essential and harmful metalloid that has
no biological benefits, pentavalent antimonials, such as
meglumine antimoniate and sodium stibogluconate (Fig. 3),
have been used for more than half a century to cure the parasitic
disease leishmaniasis, which is caused by protozoa.47 In
addition, some Sb compounds show antitumor activity.48 On
the other hand, hematuria, dermatitis, nausea, vomiting, diarrhea,
pharyngitis, and nephrotoxicity were reported to be clinical
symptoms of Sb toxicosis.49 It was also reported that Sb workers
ANALYTICAL SCIENCES OCTOBER 2009, VOL. 25
had an increased incidence of lung cancer.50 However, the
molecular mechanisms of the pharmacological and toxicological
effects of Sb are still being investigated. Recently, it is suggested
that pentavalent Sb acts as a prodrug and is converted into the
active and more toxic trivalent Sb in the body.47 Hence, the
speciation of Sb species may be required to reveal the precise
Fig. 3
Structures of Sb-containing metabolites and medicine.
Fig. 4
1193
mechanisms underlying the biological, pharmacological, and
toxicological effects of Sb.
4·2 Metabolism of antimony based on the characterization
of the antimony species by speciation studies
When rats were administered sodium antimonite, Sb was
predominantly distributed into RBCs.22,51 The accumulation in
RBCs was also observed when Te or arsenic was ingested. As
mentioned above, the accumulated species of Te and arsenic
were the dimethylated forms.45,46 To determine the chemical
species of accumulated Sb in RBCs, speciation studies were
performed. Accumulated Sb in rat RBCs was specifically bound
to hemoglobin-like DMAs and dimethylated Te.22 To specify
the chemical form of Sb bound to hemoglobin, Sb was cleaved
off from hemoglobin with hydrogen peroxide, and then the
cleaved Sb species was subjected to HPLC-ICP-MS. The
cleaved Sb compound showed the same chromatographic
behavior as that of antimonate,22 suggesting that the Sb that
specifically bound to hemoglobin was an inorganic form, not a
methylated form. Therefore, antimonite administered to rats
was directly incorporated into RBCs without methylation, and
specifically bound to hemoglobin. It is known that hemoglobin
has a specific DMAs binding site. Although it is not clear
whether or not the DMAs binding site of hemoglobin is identical
to the dimethylated Te or inorganic Sb binding site, hemoglobin
specifically binds dimethylated Te and Sb. Rodents show large
species differences in terms of tolerance to arsenic toxicity.52
The rat is the most tolerant species because it accumulates
arsenic in RBCs to reduce distribution in tissues.44 This suggests
that Sb also shows species differences among rodents.
Two Sb species were detected in rat urine when sodium
antimonite was administered at a dose of 2.0 μmol/kg body
weight.22 The retention time of one peak matched that of
antimonate (Sb(V)). On the other hand, the other peak could
not be assigned. It was reported that Sb(V) was sequestered by
some organic acids, such as citric acid, in rat urine. These
results suggested that the urinary excretion pathway for Sb
metabolism in rats was oxidation when antimonite (Sb(III)) was
administered (Fig. 4). The Sb metabolites in urine collected
Proposed metabolic pathway of Sb compared with that of arsenic.
1194
from workers of a lead battery manufacturer were also reported.53
Those workers were exposed to antimony trioxide and stilbene
(SbH3). The urinary Sb concentration of the exposed workers
was approximately 100-times higher than that of non-exposed
persons. In the urine of the exposed workers, Sb(V) was the
predominant chemical form of Sb. This suggested that oxidation
was also the main metabolic pathway of Sb in human. In the
literature, the second-most abundant metabolite was
trimethylantimony dichloride (TMSbCl2),53 suggesting that Sb
methylation occurred in human despite the fact that it was not
observed in rat. Since the rat has a higher methylation capacity
for arsenic than other mammals, it seems straightforward that
Sb would be more efficiently methylated in rat than in humans.
However, those experiments showed results that ran counter to
the speculation. Indeed, DMAs is the predominant urinary
metabolite in rat when inorganic arsenicals are ingested, whereas
monomethylated arsenical (MMAs) is detected in amounts
comparable to those of DMAs in humans who drink water
contaminated with arsenic.54,55 Only TMSbCl2, i.e., a highly
methylated metabolite, was detected as a methylated species in
human urine, suggesting that humans may have a high Sb
methylation capacity. Further studies are needed to explain this
phenomenon.
To summarize, the major metabolic pathway of Sb is oxidation
in human and rat, and methylation also occurs as a minor
metabolic pathway in human. Sb(III) is known to have ten
times higher toxicity than Sb(V). Contrary to arsenic metabolism,
Sb metabolism is simple; however, it seems that animals
functionally detoxify Sb(III) by oxidizing it to Sb(V).
5 Conclusions and Perspectives
Speciation with elemental (ICP) and molecular (ESI) MS
coupled with separation techniques has improved our
understanding of the metabolic pathways of exotic metalloids.
However, the results obtained by speciation have raised new
questions regarding the metabolism. For instance, why is
tellurosugar not biosynthesized, whereas selenosugar is a
major metabolite? What enzymes discriminate Te from Se in
selenosugar biosynthesis? Why is Sb is less methylated in rat
whereas arsenic is efficiently methylated to reduce toxicity?
Are there species differences in Sb toxicity among rodents?
We may not be able to reach the answers only by speciation.
Multi-disciplinary approaches that combine speciation,
molecular biology, and cell biology will be needed to answer
those questions and to pave the way to toxicometallomics.
6 Acknowledgements
I would like to acknowledge a Grant-in-Aid from the Ministry
of Education, Culture, Sports, Science and Technology, Japan
(No. 19390033), the Environmental Technology Development
Fund from the Ministry of the Environment, Japan, and the
financial support from Agilent Technologies Foundation, USA.
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