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Arsenic Toxicology: Five Questions
†
H. Vasken Aposhian, and Mary M. Aposhian
Chem. Res. Toxicol., 2006, 19 (1), 1-15 • DOI: 10.1021/tx050106d
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Chemical Research in Toxicology is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
JANUARY 2006
VOLUME 19, NUMBER 1
© Copyright 2006 by the American Chemical Society
ReViews
Arsenic Toxicology: Five Questions†
H. Vasken Aposhian* and Mary M. Aposhian
Department of Molecular and Cellular Biology, The UniVersity of Arizona, Life Sciences South, Room 444, P.O.
Box 210106, Tucson, Arizona 85721-0106
ReceiVed April 19, 2005
Contents
1. Preface
2. Introduction
3. Question One: What Enzyme Is Responsible
for the Methylation of Arsenic Species in the
Human?
3.1. Arsenic Biotransformations
3.2. Dual Enzymes for the Same Step in
Arsenic Biotransformation
3.3. Hydrogen Peroxide and Arsenic
Biotransformation
3.4. Conclusion One
4. Question Two: How Does Inorganic Arsenic,
More Specifically Arsenite, Inhibit the Pyruvic
Acid Dehydrogenase Multienzyme Complex?
4.1. Conclusion Two
5. Question Three: What Are the Relationships as
Judged by Urinary Arsenic Species between
Genetic Polymorphisms and Arsenic
Biotransformation in the Human?
5.1. hGSTO
5.2. hGSTO Polymorphisms
5.3. CYT 19 Polymorphisms
5.4. PNP Polymorphisms
1
2
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4
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5
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7
* To whom correspondence should be addressed. Tel: 520-621-7565.
Fax: 520-621-3709.
† This paper is written in honor of the 90th birthday of Professor John
P. Lambooy, the graduate school mentor of one of the authors. His versatility
and outspoken love for clear thinking, hard work, and original research
have always inspired the authors. The inspiration, respect, and gratitude
have remained for over 50 years.
5.5. Other Polymorphisms
5.6. Conclusion Three
6. Question Four: Is There a Useful Treatment
for Arsenic Intoxication that Can Replace BAL
(Dimercaprol)?
6.1. Treatment of Acute Exposure
6.2. Treatment of Chronic Exposure
6.3. Conclusion Four
7. Question Five: What Is the Role of Protein
Binding in Arsenic Metabolism and Toxicity?
7.1. Hemoglobin Binding
7.2. Metallothionein Binding
7.3. Other Pertinent Proteomic Papers
7.4. DIGE
7.5. Conclusion Five
8. Areas of Concern and Conclusions
8.1. Tolerance
8.2. Inappropriate Procedures for Synthesis
8.3. Inappropriate Procedures for Urine
Collection
8.4. Compound Identification
8.5. Inadequacy of the Rat as a Model for
Arsenic in Humans
8.6. Summary
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1. Preface
We have addressed five questions dealing with arsenic
toxicology: biotransformation, reactive oxygen species (ROS),1
10.1021/tx050106d CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/10/2005
2 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
polymorphism, treatment, and protein binding. The first question, “What enzyme is responsible for the methylation of arsenic
species in the human?”, still needs further investigative effort
because arsenic methylation is an important biotransformation
pathway in the human and some animals. No arsenic methyltransferase has been isolated from surgically removed or
biopsied human tissue. For CYT 19 to be accepted as the
methylation enzyme, purification of the protein and its activity
from human tissue is required. This has not been accomplished.
There is little doubt, however, that CYT 19 and the rabbit arsenic
methyltransferase have some role in arsenic biotransformation.
Second, how does inorganic arsenic, more specifically
arsenite, inhibit enzymes, e.g., the pyruvic acid dehydrogenase
(PDH) multienzyme complex? The classical mechanism is now
in doubt. The conventional belief that arsenite inhibits PDH and
perhaps other dithiol-containing enzymes by chelating or
complexing the thiol groups now needs further study because
experiments have demonstrated that PDH is more sensitive to
inhibition by ROS than to arsenic-containing agents that bind
vicinal thiols (for example, phenyldichloroarsine). ROS can be
generated by arsenicals.
Third, what are the relationships as judged by urinary arsenic
species between genetic polymorphisms and inorganic arsenic
biotransformation? It is well-accepted that there is intraindividual
variation in response to arsenic exposure as judged by the
urinary arsenic profile. Is interindividual variation due to genetic
polymorphisms? A number of polymorphisms in human GST
omega (ω), CYT 19, and purine nucleoside phosphorylase (PNP)
have been reported. Two studies have linked these polymorphisms with changes in urinary arsenic species in the human.
There have been a minimum of investigations combining studies
of polymorphisms of human genes known to be involved in
arsenic metabolism with determinations of urinary arsenic
species. In fact, the genetics of arsenic toxicity is a barren field
at present.
Fourth, is there an effective treatment for arsenic intoxication
that can replace British anti-Lewisite (BAL, dimercaprol)? meso2,3-Dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1 sulfonic acid, Na salt (DMPS) are effective in mobilizing
the excretion of arsenic from the human. DMPS seems to be
more consistently effective in the clinical improvement of
individuals chronically exposed to arsenic. With the millions
of people now known to be consuming toxic amounts of arsenic
in their drinking water or food, a large-scale clinical trial of
arsenic antidotes is needed and recommended. An effective
arsenic-mobilizing agent is of little benefit as long as exposure
continues but may be of value to decrease the arsenic body
burden once the exposure has ceased.
Fifth, what is the role of protein binding in arsenic metabolism
and toxicity? Are proteins only the site of the toxic action of
arsenic species or are they also an arsenic-binding storage
reservoir involved in detoxication? A recent study clearly has
shown that the differences in the number of cysteine residues
in human and mouse hemoglobin are responsible for the greater
accumulation of arsenic species in rat blood. With new
1Abbreviations: DMA, generic term including DMA(III) and DMA(V); DMA(III), dimethylarsinous acid; DMA(V), dimethylarsinic acid; DTT,
dithiothreitol; MMA, generic term including MMA(III) and MMA(V);
MMA(III), monomethylarsonous acid; MMA(V), monomethylarsonic acid;
SAM, S-adenosyl-methionine; GST, glutathione-S-transferase; PDH, pyruvate dehydrogenase; PNP, purine nucleoside phosphorylase; ICP-MS,
inductively coupled plasma mass spectrometry; BAL, British anti-Lewisite;
DMSA, meso-dimericapto succinic acid; DMPS, 2,3-dimercaptopropane1-sulfonic acid, Na; PAO, phenylarsineoxide; ROS, reactive oxygen species;
hGSTO, human glutathione-S-transferase ω; EST, expressed sequence tags;
DIGE, differential in-gel electrophoresis.
Aposhian and Aposhian
Scheme 1
proteomic techniques available, such as differential in-gel
electrophoresis (DIGE), research in arsenic toxicology now may
be expanded to acquire more specific knowledge as to the exact
role of specific proteins in arsenic intoxication and detoxication.
Two-dimensional electrophoresis procedures, in which protein
extracts from two subjects can be electrophoresed together and
simultaneously by using two different fluorescent dyes, is a
productive approach to help answer these questions.
While a number of recent papers have been emphasized in
this review, the need for confirmation of their conclusions by
other investigators is needed. The importance of their results at
this time, however, should not be minimized. Rather, they should
stimulate investigators to reexamine and expand their thinking
and investigations and, hopefully, attract new investigators.
2. Introduction
According to ancient Greek myths, Hercules killed Hydra
and then dipped all of his arrows into the poisonous venom of
Hydra’s many arms. Subsequently, Hercules used these poisoned
arrows to win his legendary battles to atone for his wrongdoings.
The word for arrow in ancient Greek is toxin. Thus, the names
toxin and toxicology can be traced back to stories of Hercules
and the poison-filled, many-armed Hydra. Figuratively, the
toxicology of inorganic arsenic also has many arms (Scheme
1). Many of these will not be addressed in this review because
it is not meant to be a summary of the literature. We do not
feel compelled to cite every arsenic toxicology paper published
during some given time period. Rather, the purpose of this
review is to deal with some of these arms by addressing five
important questions central to arsenic toxicology with the intent
of stimulating investigators, especially new ones, to seek
answers.
First, what enzyme is responsible for methylation of arsenic
species in the human? Second, how does inorganic arsenic, more
specifically arsenite, inhibit enzymes, e.g., the PDH multienzyme
complex? The classical mechanism is now in doubt. Third, what
are the relationships as judged by urinary arsenic species
between genetic polymorphisms and inorganic arsenic biotransformation? Is interindividual variation due to polymorphisms?
Fourth, is there an effective treatment for arsenic intoxication
that can replace BAL (dimercaprol)? Fifth, what is the role of
protein binding in arsenic metabolism and toxicity? Are proteins
ReViews
Figure 1. Generally accepted pathway for biotransformation of
inorganic arsenic. Is it becoming obsolete?
Figure 2. Proposed pathway by Hayakawa et al. (5) for biotransformation of inorganic arsenic. Reprinted with permission from ref 5.
Copyright Springer Science and Business Media.
not only the site of the action of toxic arsenic species but also
an arsenic-binding storage reservoir involved in detoxication?
Only the papers that we consider to be pertinent to these five
questions have been cited. Carcinogenicity, signal transduction,
and the use of arsenic trioxide to cure human cancer are not
reviewed because each could be the single topic of a review
and many other investigators are much more competent and
knowledgeable in such areas (1-4).
3. Question One: What Enzyme Is Responsible for the
Methylation of Arsenic Species in the Human?3.1.
Arsenic Biotransformations
First, a review of inorganic arsenic biotransformation is
pertinent. The usually accepted pathways from inorganic arsenate to dimethylarsinate are outlined in Figure 1. Although a
figure very often is interpreted as a final version embedded in
stone, the authors wish to make it clear that there are many
uncertainties and unknowns about this one. This can be seen in
comparing the reactions of Figure 1 with a very interesting and
novel pathway for inorganic arsenic biotransformation (Figure
2) suggested by Hayakawa et al. (5).
The generally accepted pathway of arsenic biotransformation
(Figure 1), usually credited to Challenger (6) and Cullen and
Reimer (7), consists of a series of reductions and oxidations
coupled with methylations (Figure 1). In the reactions, the +5
oxidative arsenic species is formed before the analogous +3
arsenic species e.g., monomethylarsonic acid [MMA(V)] before
monomethylarsonous acid [MMA(III)], dimethylarsinic acid
Chem. Res. Toxicol., Vol. 19, No. 1, 2006 3
[DMA(V)] before dimethylarsinous acid [DMA(III)], and
TMAO before TMA(III). Only two putative enzymes, MMA(V) reductase and arsenic methyltransferase, in the pathway have
been extensively purified and studied (8-13) as has an
alternative one, CYT 19 (14-18). Human MMA(V) reductase
and hGST ω are identical proteins (13). Both names are used
in this paper to allow a greater relevance and emphasis as needed
for the reader.
The recent Hayakawa et al. proposal (5) appears to be a
reasonable pathway for arsenic biotransformation. The most
original part of their proposal is that +3 arsenic species are
formed before +5 species, the former being oxidized by
hydrogen peroxide or other agents to produce +5 species that
are end products of arsenic metabolism. At least one of them,
DMA(V), has been the major end product found in the urine of
most species and in the past believed to be the end point of
arsenic metabolism after exposure.
The Hayakawa et al. (5) model (Figure 2) proposes that
arsenic triglutathione (ATG) and monomethylarsonic diglutathione [MA(SG)2] are substrates of CYT 19, one possible
methylating enzyme. This new pathway also opens the possibility that these glutathione-arsenic compounds, in order to
be formed and oxidized, need new enzymes. Once the arsenicglutathione substrates are formed, it is proposed that they are
methylated by CYT 19 and S-adenosyl-methionine (SAM) to
form MADG and DMAG. The methylated compounds are then
oxidized to MMA(V) and DMA(V), the major arsenic metabolites found in the urine. Aposhian et al. (19, 20) in 2004
proposed that the more toxic +3 arsenic species might be
oxidized and detoxified by hydrogen peroxide to form the less
toxic +5 species. The differences between this newly proposed
pathway (Figure 2) and the older one (Figure 1) are important,
although the urinary arsenic species, namely, the +5 species,
are the same. What is most attractive about the Hayakawa
scheme is that MMA(V) and DMA(V) are proposed as the end
products, not just intermediates, in arsenic biotransformation.
The Cullen and Reimer (7) scheme had the anomaly of the major
intermediates appearing as end products in the urine.
These experiments of Hayakawa et al. (5) are important and
novel enough to require confirmation. One of the major
criticisms of its proposed pathways is that the structures of the
new intermediates involved, specifically the arsenic-glutathione
substrates and products, have not been confirmed by adequate
procedures. Chromatography retention times are no longer
acceptable as the only proof of structure of new, closely related
arsenic compounds. Second, a search of the scientific literature
indicated that human CYT 19 has been produced only by DNA
recombinant technology (18). It has not been purified and
isolated from human tissue. To suggest that human CYT 19 is
the arsenite methyltransferase, when CYT 19 does not appear
to be expressed in human liver, is at present unwarranted. A
search of NCBI (21) indicated that CYT 19 mRNA does not
appear to be expressed or is expressed in only small undetectable
amounts in human liver (Figure 3). The expression profile
suggested by analysis of expressed sequence tags (EST) events
in human liver was zero transcripts per million RNA molecules.
In mouse liver, it was 76 transcripts per million RNA molecules
(21). It does appear to be expressed in human kidneys and a
few other tissues. Yet, the major site of arsenic methylation
always has been claimed to be the liver (22, 23). However, in
a recent letter to us, Dr. David S. Barber of The University of
Florida has informed us that he has “observed expression of
CYT 19 at the message level using RT-PCR in 8 of 8 human
liver samples from male Caucasians” (personal communication).
4 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Aposhian and Aposhian
Figure 3. mRNA transcripts of human CYT 19. Expression profile suggested by analysis of EST counts (21). The National Library of Medicine
Web pages are public domain.
While this is definitely a step forward, the enzyme activity
responsible for methylating inorganic arsenic in humans still
has not been purified from human tissue. Neither have there
been reported experiments to determine whether CYT 19 is
inducible. Experiments in our laboratory dealing with the
inducibility of rabbit methyltransferase have been numerous but
inconclusive.
3.2. Dual Enzymes for the Same Step in Arsenic
Biotransformation
It is pertinent to this review to examine the hypothesis that
alternative reactions are available for each step of the biotransformation of inorganic arsenic beginning with the reduction of
arsenate to arsenite. For many years, it was proposed that this
reduction was accomplished by GSH in a strictly chemical,
nonenzymatic reaction. Radabaugh and Aposhian (24) showed
that extracts of human liver carried out the reduction of arsenate.
In 2002, Radabaugh et al. (10) reported that PNP with inosine
as a required constituent of the enzyme reaction had arsenate
reductase activity. Simultaneously, the Gregus group (25)
reported the same reaction in rat tissue. Subsequently, the latter
group reported that PNP was not a factor in arsenate reduction
(26) but their studies can be criticized for using the rat, a species
generally acknowledged to be a poor model for inorganic arsenic
metabolism in the human (see the discussion of question five
below) and ignoring the functions of hGSTO (8, 13).
The primary reaction for arsenate reduction in humans is
catalyzed by human glutathione-S-transferase ω (hGST-01) (8,
13, 27-29) and is probably the explanation for a process
described in a paper by other workers dealing with the GSHdependent reduction of arsenate in human erythrocytes (26).
The Km and Vmax data for hGSTO1 are given in Table 1. The
enzyme requires GSH.
Once arsenate is reduced to arsenite, it is methylated. An
enzyme from rabbit liver or a human hepatocyte cell line has
Table 1. Kinetics Data for hGSTO1-1 (27, 28)
substrate
Km (M)
Vmax (µmol/mg/h)
arsenate
MMA(V)
DMA(V)
34.8 × 10-3
53.6 × 10-3
30.6 × 10-3
12.8
52.6
18.0
been partially purified that will catalyze the methylation of
arsenite or MMA(III) (9, 11, 12, 29). SAM is the methyl donor.
Because a reducing agent such as GSH or L-cysteine is needed
for methylation, the glutathione-arsenic type structures proposed by Hayakawa et al. (5) as substrates or intermediates are
attractive. Certainly, the methylation of arsenite or MMA(III)
in vitro can be carried out by either the rabbit type of
methyltransferase (11), found in a variety of animals including
the hamster (12) and lacking in the chimpanzee, tamarin, and
guinea pig, or the rat CYT 19 methyltransferase (14).
The CYT 19 relevance has been enormously strengthened
by the preparation of human CYT 19 using DNA recombinant
technology (18). In addition, it has been suggested recently that
the lack of arsenic methylation in the chimpanzee is due to a
275 nucleotide deletion in its CYT 19 gene beginning at
nucleotide 612 leading to a premature stop codon (18). In no
way does the present discussion mean to minimize the importance of CYT 19, especially in the rat. Its importance and
relevance to the methylation of arsenic species in general are
established. The rabbit methyltransferases are not without
problems. Although extracts have been purified 9000-fold (30),
sequence identification has been unsuccessful.
Thus, we have two alternative methylation and three reduction
pathways for inorganic arsenic metabolism. Scheme 1 uses GSTω, PNP, or perhaps GSH nonenzymatically for arsenate reduction, arsenite/MMA(III) methyltransferase or CYT 19 for
methylation, and GSTO for subsequent reduction of methylarsenic(V) and dimethylarsonic(V) species. On the other hand,
the Hayakawa et al. (5) scheme uses GSH complexed with +3
ReViews
Chem. Res. Toxicol., Vol. 19, No. 1, 2006 5
arsenic species as substrates for methylation to +3 arsenic
species and perhaps oxidation by H2O2 to resulting +5 species.
It should be understood that the biotransformation of inorganic
arsenic in many animals and perhaps the human needs only two
enzymes, hGSTO and a methyltransferase. The attempts to link
other reducing enzymes and systems are unnecessary in our
opinion. Perhaps some investigators would be helped by
remembering and utilizing Occam’s razor (31).2 On the other
hand, as compared to many other complex biological structures,
arsenate has a relatively simple structure that is composed
primarily of an arsenic atom and oxygen atoms. It should not
be surprising that other reducing enzymes might reduce arsenate
to arsenite.
3.3. Hydrogen Peroxide and Arsenic
Biotransformation
The importance of hydrogen peroxide is often underestimated
in toxicology. It is usually considered a dangerous oxidant that
can damage the structure of the DNA, RNA, and proteins in
the cell (32). It is also, however, an important signal that by
reacting with thiols of proteins can turn on (or off) signaling
pathways. Human peroxiredoxins rid the cell of H2O2 faster than
other antioxidant enzymes and are considered to be the
controlling switch that determines whether H2O2 is to be a
damaging agent or a signaling agent (32). To all of this now
must be added the possible role of hydrogen peroxide in the
metabolism of inorganic arsenic where its oxidation role is
becoming more evident. There also are signals being sent by
the H2O2 to other biological processes involving arsenic (33).
Before we leave this discussion of arsenic biotransformation,
it is pertinent to ask: Why does DMA(V) especially and to
some extent MMA(V) appear in human urine in large concentrations? Although water solubility has been cited by some to
be the reason, a more reasonable explanation is that DMA(V)
and MMA(V) are much less reactive and have much less affinity
for tissue components than do the more reactive DMA(III) and
MMA(III). Also, there is a stability problem with MMA(III)
and DMA(III), which can be easily oxidized. Finally, the
Hayakawa et al. (5) scheme has proposed MMA(V) and DMA(V) as the end points of metabolism, not as intermediates (Figure
2).
3.4. Conclusion One
As to the question of what enzyme catalyzes the methylation
of arsenic in the human, the answer is still unknown. There are
the rabbit type methyltransferases and the CYT 19. The
relevance and importance of each are established, but questions
about both remain, especially for the human. Until an enzyme
is purified from surgically removed human tissue, the question
remains unanswered. Perhaps more novel approaches in studying
arsenic biotransformation are needed. The Hayakawa et al. (5)
scheme introduces new reactions especially for the formation
as well as the oxidation of arsenic-glutathione compounds.
Investigations of enzymes involved in these new pathways are
needed.
4. Question Two: How Does Inorganic Arsenic, More
Specifically Arsenite, Inhibit the Pyruvic Acid
Dehydrogenase Multienzyme Complex?
For many years, the usually accepted mechanism for arsenite
toxicity has been that it combines with thiols, especially the
2I think the following is the most useful statement of this principle for
scientists: “When you have two competing theories which make exactly
the same predictions, the one that is simpler is better.”
vicinal thiols of enzymes, and this was believed to result in the
inhibition of the catalytic activity (34-36). One of the most
studied enzyme models has been the pyruvate dehydrogenase
(PDH) complex. It has been considered the target site most
sensitive to inhibition by arsenite. More specifically, the site
has been believed to be the lipoamide dehydrogenase subunit
of the large PDH multienzyme complex (37, 38). PDH catalyzes
the oxidative decarboxylation of pyruvate to form acetyl CoA.
Inhibition of PDH would be expected to disrupt the energy
system of the cell with resulting cell damage and death.
Recently, Samikkannu et al. (39) asked the question: Does
arsenic trioxide inactivate PDH activity in human cells via
binding to the thiols of the enzyme complex or via inhibition
by ROS? The results of this provocative and important paper
indicated that arsenic trioxide exposure stimulates ROS production that causes PDH inactivation by oxidation. These results
have disrupted the dogma as to the mechanism of inorganic
arsenic inhibition of enzymes and other metabolic processes.
They compared the PDH activity of HL60 cells and purified
porcine PDH after exposure to either arsenic trioxide or
phenylarsineoxide (PAO). Surprisingly, the intracellular PDH
activity, measured in extracts after the cells had been exposed
to arsenic trioxide, was more sensitive to inhibition than purified
porcine PDH. The IC50 values were 2 and 182 µM, respectively.
Arsenic trioxide was approximately 90 times more inhibitory
for the intracellular PDH than for the purified PDH. This is
quite unusual since purified enzymes are usually more sensitive
than cells to inhibitors because the latter has membranes to be
penetrated, stimulators, inhibitors, and/or other interfering
substances.
Phenylarsineoxide reacts specifically with vicinal thiols of
proteins to form stable rings. Models for the inhibition of lipoic
acid-containing enzymes by PAO have appeared (37, 38). When
PAO and arsenic trioxide were compared as to their activity as
thiol and vicinal thiol-reacting agents, PAO was more potent
(39). The IC50 values for PAO to decrease the thiol and vicinal
thiol content of HL60 cells were 2.3 and 1.9 µM, respectively,
while for arsenic trioxide they were 82.5 and 81.7 µM,
respectively. It is pertinent to point out that the IC50 value for
arsenic trioxide to inhibit HL60 PDH activity was 2 µM, a level
found in humans after inorganic arsenic exposure, while the
IC50 value of arsenic trioxide to decrease thiol and vicinal thiol
content of HL60 cells was about 80 µM.
Dithiols such as dithiothreitol (DTT), DMSA, or DMPS
prevented the PAO inhibition of PDH activity in HL60 cells,
but at the level used, they did not reverse the arsenic trioxidecaused inhibition. However, antioxidants such as pyruvate,
catalase, and selenite prevented the arsenic trioxide inhibition
of PDH but not the PAO inhibition. Arsenic trioxide, but not
PAO, increased hydrogen peroxide levels in the HL60 cells.
Thus, according to the Samikkannu et al. (39) results, arsenite
exposure appears to increase hydrogen peroxide production,
producing hydroxyl free radicals through the Fenton reaction
resulting in oxidative damage and inactivation of the PDH
protein. This process occurs at arsenite concentrations much
lower than that for arsenite binding to critical and essential thiols.
The production of ROS in biological systems after arsenic
exposure is well-known (40, 41). Oxidation of toxic +3 arsenic
species by hydrogen peroxide has been proposed as a mechanism for decreasing their toxicity (19, 20). These innovative
studies by Samikkannu et al. (39) are very provocative. They
dealt, however, primarily with a human leukemia cell line. The
extension of these experiments to nonleukemic cells and whole
animals might further strengthen their hypothesis. A careful
6 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Aposhian and Aposhian
reading of Samikkannu et al. (39) is recommended for new
insights dealing with the inhibitory properties of inorganic
arsenic. In addition, the Samikannu et al. (37) proposal is
strengthened by the study in humans that showed that oxidative
stress could be caused by chronic exposure of humans to
drinking water containing inorganic arsenic (42). Elevated serum
lipid peroxide levels and decreased nonprotein sulfhydryl were
correlated with blood levels of arsenic species (42).
4.1. Conclusion Two
“How does inorganic arsenic, more specifically arsenite,
inhibit the pyruvic acid dehydrogenase multienzyme complex?”
The well-entrenched, usually accepted, mechanism that inhibition by arsenite is the result of its reaction with PDH vicinal
thiols is now under question. The generation of ROS by arsenite
and the resulting oxidative damage to proteins (PDH) is a
reasonable mechanism for the inhibition of PDH by arsenite.
This novel and reasonable explanation for arsenite mitochondrial
toxicology involving ROS production and inhibition deserves
confirmation and further study.
5. Question Three: What Are the Relationships as
Judged by Urinary Arsenic Species between Genetic
Polymorphisms and Arsenic Biotransformation in the
Human?
Recently, genetic techniques have been applied to this
problem. According to one of the biotransformation pathways,
only two genes and enzymes are necessary for the metabolism
of inorganic arsenic in the human, hGSTO and an arsenic
methyltransferase. It appeared that the most relevant gene and
enzyme to study in order to unravel the cause of interindividual
variability of arsenic metabolism in the human was hGSTO.
Its gene, protein structure, and amino acid sequences were
known (12, 13, 43). Its properties and its relationship to the
metabolism of arsenic by humans were understood (8, 13, 43).
Figure 4. Speciation of urinary arsenic as a percentage of total
inorganic arsenic (52). Also included are the averages of all of the
subjects from La Virgen and the average of all 75 subjects in the study
population. The analysis did not detect MMA(III) and DMA (III) for
reasons unknown to the investigators.
Table 2. Concentration of Arsenic Species in the Urine (µg/g
Creatinine) (52)a
subject no.
As(V) As(III) MMA(V) MMA(III) DMA(V) DMA(III)
44
132.6
6.2
47
4.5 244.9
average for
5.1
14.4
100 µg As/L
group
average for all
7.91 16.02
groups, n ) 75
a
7.1
9.3
9.7
ND
ND
0.18
14.1
0.43
29.7
114.2
54.5
81.17
NDa
ND
ND
ND
ND, not detected.
hGSTO-1 and hGSTO-2, the dehydroascorbate reductase specific activity of hGSTO-2 was 70-100 times greater than that
of hGSTO-1 (43, 48, 51).
5.2. hGSTO Polymorphisms
5.1. hGSTO
A brief review of this enzyme is appropriate. MMA(V)
reductase and hGSTO-1 are identical proteins (13) that can
reduce arsenate, MMA(V), and DMA(V) to arsenite, MMA(III), and DMA(III), respectively (27, 28). The products are more
toxic than the substrates (44, 45). No other enzyme is necessary
for the reduction of these arsenic species.
The glutathione-S-transferases (GSTs) make up a superfamily
of intensively investigated enzymes that have many different
functions. Reviews are available (46, 47). The function most
usually associated with them is the conjugation of GSH with
xenobiotics. Discovered by the Board group (43), the ω class
GSTs have many properties different from other mammalian
GSTs such as R, µ, π, and κ. The ω GSTs have thiol transferase,
dehydroascorbate reductase, as well as arsenate, MMA(V), and
DMA(V) reductase activity. The other members of the GST
superfamily essentially lack such activities (43, 48). GSTO1-1
is expressed in many human tissues as indicated by mRNA
transcription (49) and in hamster tissue as judged by enzyme
activity (50). The GST ωs have a cysteine (Cy 32) in the active
site rather than serine or tyrosine, which have been found in
other mammalian GSTs (40). GSTO2 has been recently solubilized and characterized (48). Both hGSTO1 and hGSTO2 have
six exons and are separated by 7.5 kb on chromosome 10q24.3.
There is 64% amino acid identity of hGSTO2 with hGSTO1.
Although many substrates are reduced almost equally by
The Aposhian group in collaboration with Professor Gonzalo
Garcia-Vargas studied a group of 39 females and 36 males living
in the vicinity of Durango and Torreon, Mexico (52). Most of
the subjects were chronically exposed to high levels of arsenic
in their drinking water. When 11 h overnight urines were
collected and analyzed for arsenic species by HPLC-inductively
coupled plasma mass spectrometry (ICP-MS), two of the
subjects were found to have unusual urine arsenic profiles
(Figure 4 and Table 2). Subject 44 had an unusually large
amount of arsenate, almost 76% of the total urinary arsenic,
and much lower amounts of arsenite, MMA(V), and DMA(V).
When his hGSTO1 gene was examined, polymorphisms indicated that the glutamate at 155 had been deleted and the
glutamate 208 was replaced by lysine. The other subject, 47,
also had an unusual urine arsenic profile and was the mother
of subject 44. She had an unusually large percent of arsenite in
her urine (Figure 4 and Table 2). While the glutamate 155
deletion and the 208 glutamate replacement by lysine were
common to both, the mother also had the 140 alanine replaced
by asparagine as well as a GGC deletion.
The urine arsenic profile of subject 44 might be explained
by a polymorphism resulting in an inhibition of the arsenate
reducing activity of hGSTO1. The urine arsenic profile of
subject 47 might be explained by the increased activity of
hGSTO1 and/or an inhibition of arsenic methyltransferase. Some
support for this is that in a variant protein expressed in
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Chem. Res. Toxicol., Vol. 19, No. 1, 2006 7
Table 3. Specific Activity of Allelic Variants of GSTO1-1 and
GSTO2-2 (48)a
monomethylarsonate(V) dimethylarsonate(V)
reductase
reductase
Ala 140/Glu155Glu208
Asp140/Glu155Glu208
Ala140∆Glu155/Glu208
Ala140/Glu155/Lys208
Ala140∆Glu155/Lys208
GSTO1-1
0.33 ( 0.037
0.27 ( 0.039
0.65 ( 0.007
0.39 ( 0.037
0.67 ( 0.066
0.12 ( 0.006
0.15 ( 0.005
0.30 ( 0.014
0.26 ( 0.006
0.37 ( 0.015
Asn142
Asp142
GSTO2-2
0.42 ( 0.071
0.44 ( 0.044
0.03 ( 0.012
0.05 ( 0.019
a All activities are shown as µmol NADPH consumed per min per mg
protein at 30 °C. All values are the means of at least three determinations
( standard deviation.
Escherichia coli, a deletion of glutamate 155 doubled the MMA(V) and tripled the DMA(V) reductase activity of hGSTO1-1
(48). Perhaps, the arsenate reductase activity also was increased.
Unfortunately, the arsenate reductase activity was not determined. Another variant protein in which glutamate 208 was
replaced by lysine had a minimal effect on MMA(V) reductase
activity and almost doubled the DMA(V) reductase activity
(Table 3). Other polymorphisms and genotypes also have been
found (48, 51).
The MMA(V) reductase activity of hGSTO2 was slightly
different than that of hGSTO1-1 (Table 3). The DMA(V)
reductase activity of hGSTO2-2 was about one-fourth that of
hGSTO1-1. A polymorphism for hGSTO-2 consisting of the
substitution of the 142 asparagine by aspartic acid (N142D) was
detected (48), but it was without significant effect on the specific
activity of this enzyme with its usual substrates (Table 3).
The Klimecki group (53) has studied polymorphisms in
hGSTO and PNP in 22 European and 24 indigenous American
individuals. A total of 33 hGSTO1-1 polymorphisms were
observed. The Europeans had more polymorphisms in the
hGSTO gene than did the indigenous Americans. The genetic
polymorphisms of each group were essentially exclusive to that
group. For the European group, the minor allele frequency was
34% for Ala140Asp, 5% for the glutamic 155 deletion, and 5%
for glutamate 208 lysine. These polymorphisms were absent
from the indigenous American group. On the other hand, the
indigenous American group had a 4% frequency of the
Ala236Val, which was absent in the European group. Because
the Klimecki study (53) has been the most complete one dealing
with genetic variations in hGSTO1-1, including an extensive
list of intron polymorphisms, a figure from their paper is
included for informational purposes (Figure 5).
Tanaka-Kagawa et al. (54) characterized two GSTO-1
recombinant variants. The 140 alanine was replaced by asparagine, and in the second variant, the 217 threonine was replaced
by asparagine. The former variant had similar kinetics as the
wild when MMA(V) was the substrate. The Km and Vmax values
of the Thr217Asn variant were 64 and 56%, respectively, of
the wild type, but its relevance has been questioned (48).
5.3. CYT 19 Polymorphisms
Polymorphisms for human CYT 19 have been found (55) in
a Mexican population (Figure 6). Some of these sites were
associated with the ratio of urinary dimethylarsinate:monomethylarsinate in children. Ratios, however, usually exaggerate
small differences. The authors neglected to consider and cite
the work from Vahter’s group pointing out the unusual decreased
amount of MMA in a small number of indigenous American
children (56). It seems appropriate at this time to point out that
the consequences of acute and chronic exposure to arsenic in
children are often a neglected area of arsenic research. Papers
by Concha et al. (56) and Calderon et al. (57) are recommended
reading.
5.4. PNP Polymorphisms
An investigation of PNP polymorphisms found that glycine
51 was replaced by serine in exon 2 in 14% of European group
and 35% of the indigenous American group (53). The latter
group had a greater number of PNP polymorphisms. The
relevance of PNP for arsenic metabolism has become controversial (10, 26). Although other polymorphisms were found,
they do not appear pertinent at the present time.
5.5. Other Polymorphisms
There have been other studies as to how polymorphisms in
other genes may affect arsenic metabolism, but the genes studied
were not proven to be directly involved in arsenic metabolism.
In a population from northeastern Taiwan, there was a slightly
increased percentage of urinary inorganic arsenic for the null
genotype of GSTM1 and an elevated percentage of urinary
DMA in null genotype of GSTT1 (58). A genetic polymorphism
in p53 involving a change from arg/arg to pro/pro has been
suggested to increase the risk of skin cancer in subjects in
southwest Taiwan (58).
Ironically, arsenic trioxide has been used for the successful
treatment of acute promyelocytic leukemia. In some patients,
however, there was increased toxicity and even death (59).
Polymorphisms in hGSTO or other genes involved in arsenic
biotransformation should not be overlooked as a potential cause
of these severe reactions.
5.6. Conclusion Three
To be able to decipher and ascertain the specific areas of the
genes that may be responsible for the interindividual variations
found in humans chronically exposed to inorganic arsenic, it is
advisable to measure changes in at least three parameters: gene
nucleotide sequence, the relevant enzyme activity of the gene
product, and changes in the concentrations of various arsenic
species in the urine. While it is advisable to study all of these
changes in one laboratory, it appears that very few, at present,
have all of the capabilities necessary. Polymorphisms of
hGSTO1 and CYT 19 have been correlated with some changes
in urine arsenic species, but much more research dealing with
polymorphisms of hGSTO and CYT 19 and correlating them
with the urine profiles of arsenic species is needed.
6. Question Four: Is There a Useful Treatment for
Arsenic Intoxication that Can Replace BAL
(Dimercaprol)?
Because arsenic has been used for many years as a suicidal
or homicidal agent, because it is a major contaminant of drinking
water in a number of countries (60-62), and because it is
present in Lewisite, the chemical warfare agent, it is not
surprising that there have been continuing efforts to find
antidotes for it. It should be clearly understood, howeVer, that
the best way to deal with arsenic toxicity is to preVent exposure.
BAL (dimercaprol, 2,3 dimercapto-1-propanol) (Figure 7) was
developed during World War II as an antidote against the
arsenic-containing chemical warfare agent Lewisite (34, 63).
BAL is a fat-soluble, easily oxidizable oil that must be given
8 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Aposhian and Aposhian
Figure 5. Summary of frequency and gene context of polymorphisms discovered in hGSTO1 in European ancestry (Europe) and indigenous
American (America) ancestry subjects (53). The ID column indicates the polymorphism identification numbers relative to the location in the consensus
sequence with the first base of the consensus numbered 1. The ATG offset column indicates the polymorphism location relative to the first base
“A” of the ATG methionine initiation codon. The freq % column is the minor allele frequency graphically displayed in the column to the right (53).
Reprinted with permission from EnVironmental Health PerspectiVes.
Figure 6. Summary of frequency and gene context of polymorphisms discovered in CYT 19 in European ancestry (Europe) and indigenous
American (America) ancestry subjects (55). The ID column indicates the polymorphism identification number relative to the location in the consensus
sequence, with the first base of the consensus numbered 1. The ATG offset column indicates the polymorphism location relative to the first base
“A” of the ATG methionine initiation codon. The freq % column is the minor allele frequency, graphically displayed in the column to the right
(55). Reprinted with permission from EnVironmental Health PerspectiVes.
by deep intramuscular injection. More than 50% of the patients
to whom it is administered suffer side effects. While the side
effects usually are not serious and quickly disappear when BAL
treatment is stopped, they are annoying and discomforting. In
addition, because it is not given by mouth, because of its
instability during storage, and because of concerns that treatment
with it resulted in increased brain arsenic levels in rabbits (64),
BAL has been replaced by two water-soluble chemical analogues, DMPS and DMSA. The former was synthesized and
tested in animals by Petrunkin’s group in Kiev (65). DMSA
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Chem. Res. Toxicol., Vol. 19, No. 1, 2006 9
Figure 7. Chemical formulas for chelating agents used to treat arsenic toxicity.
was developed by Ding and Liang (66) in China and Freidheim
(67) in Switzerland. The latter agent is approved by the U.S.
Food and Drug Administration (FDA) for treating children with
blood lead levels equal to or greater than 45 µg/dL. In addition,
there have been a number of “off-label” uses as arsenic and
mercury antidotes (68).
Although no drug is without unwanted side effects, DMPS
is a relatively safe chelating and reducing agent approved in
Germany for treatment of mercury toxicity. However, there have
been reports of serious idiosyncratic reactions to it (69). Even
though a new drug application for it has never been submitted
to the U.S. FDA, it has been used extensively in the United
States by alternative medicine physicians who obtain it from
compounding pharmacists. An excellent, extensive, well-written
monograph entitled “Dimaval, DMPS” (70) is available from
the manufacturer and its U.S. subsidiary Heyl-Tex. The present
authors recommend that any physician prescribing DMPS obtain
and read it.
6.1. Treatment of Acute Exposure
After two brothers ingested 1 and 3 g of pure arsenic trioxide,
DMPS was given intravenously at 5 mg/kg every 4 h for 24 h
and then 400 mg orally every 4 h for an additional 5 days. There
was no prolonged renal or neurological impairment that usually
is seen in untreated arsenic poisoning (71). There have been
other reports of the successful use of DMPS for recovery from
arsenic-induced neuropathy (72).
A recent report (69) concerning a young man who tried to
commit suicide by ingesting arsenic trioxide is of interest. The
concentration of total arsenic in the first urine collected after
hospital admission was 215 mg/L. After 8 days of DMPS
treatment, it decreased 1000-fold. The chelating agent was
administered over 12 days with a total dose of 15.25 g of DMPS,
some by iv perfusion and some by mouth. The urinary DMA
accounted for less than 5% of total urinary arsenic. It usually
accounts for 60-70%. On day eight of therapy, the urine
contained arsenite, arsenate, MMA, and DMA, the sum of which
amounted to only 64.4% of the total urinary arsenic indicating
that the excreted DMPS-arsenic species complex was not being
detected or measured by the analytical method employed. This
had been noted and suggested previously in studies by the
Aposhian group (74-76). The almost complete absence of DMA
state may have been due to the inhibition of the second
methylation reaction, although other mechanisms are possible
(76).
6.2. Treatment of Chronic Exposure
Treatment after chronic exposure to toxic levels of inorganic
arsenic, usually via drinking water but also by the ingestion of
food contaminated by coal fly ash containing large amounts of
arsenic (77), has presented a challenge. To decrease the body’s
arsenic burden and then return the patient to the area where
arsenic exposure occurred is of little and questionable benefit
to the patient. Both DMPS and DMSA have been studied.
DMPS increased the urinary excretion of arsenic in such chronic
exposures (78, 79), but just because the excretion of a toxic
metal has been increased does not mean clinical improvements
always occur (80). Guha Mazumder et al. (81), using a
randomized placebo-controlled trial of DMPS and a scoring
system before and after treatment, concluded that this chelating
agent not only increased urinary excretion of arsenic but also
improved the clinical score that was used to judge the clinical
condition of the arsenosis patients. The number of patients, 11,
was small. Although DMSA has had success in increasing the
urinary excretion of arsenic after acute exposure (73), it was
unsuccessful in reversing the biochemical or histological
response in chronic arsenosis (83).
While a clinical trial with a much larger number of subjects
needs to be done, the immediate usefulness of such a trial for
people in India and Bangladesh is doubtful since after chelation
treatment most would return to their homes where exposure
would continue. Thus, the chelation treatment would appear to
have limited, if any, justification. In addition, chronic arsenosis
occurs in areas that are among the poorest in the world, for
example, Bangladesh, the West Bengal region of India, southwest China, Mongolia, and others. Most people in these areas
could not afford chelation therapy, if it were useful and available.
We cannot help but wonder in this time of concerns over
environmental justice if chronic arsenic toxicity due to drinking
contaminated water had occurred in the developed, wealthy,
Western countries rather than in the poor, developing countries,
that an inexpensive, easy to use water purification procedure
as a solution to the greatest public health calamity of the last
30 years would have been achieved by now. The economic
middle and upper classes can buy low arsenic bottled water.
The poor cannot, and it is their children who suffer the most
from such environmental injustice. Excellent and more extensive
reviews of chelating agents by Andersen (84) and BAL by
Muckter et al. (85) are available and highly recommended for
reading.
In unexposed individuals, the arsenic concentrations usually
found in the blood are 2.5 µg As/L, and in the urine, they are
10-50 µg As/L (82). A recent report concerning the concentrations of arsenic species in human organs after arsenic trioxide
poisoning is available and useful (86).
It appears reasonable that a cheap, effective, and useful
method of purifying highly contaminated drinking water eventually will be found. At that time, the removal of body stores of
arsenic needs to be considered. The removal of these arsenic
10 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
storage depots may be necessary to eliminate any potential
carcinogenic manifestations that had not occurred up to that
time. It is at such a point that chelating agents might be helpful.
6.3. Conclusion Four
Although the clinical studies are limited, it would appear that
DMPS is the best drug available for increasing the excretion of
arsenic and improving the conditions of humans exposed to
various forms of this metalloid. Idiosyncratic reactions, however,
have occurred. A well-designed, large clinical trial of the
effectiveness of DMPS in treating chronic arsenosis is needed.
It is necessary to plan now for what will be needed once a useful
method for purifying drinking water contaminated with high
concentrations of arsenic becomes available. At that time, there
may be the need to use DMPS or other chelating agents to
reduce body stores of arsenic to minimize arsenic cancer risks.
7. Question Five: What Is the Role of Protein Binding
in Arsenic Metabolism and Toxicity?
A number of early reports appeared dealing with the binding
of arsenic species to proteins (87-90). The importance of such
binding is at least 2-fold. First, it includes the binding and
resulting inhibition of enzymes to cause arsenic toxicity. Second,
it may be a mechanism for the modulation of arsenic toxicity
by immobilization of the arsenic compound in an arsenic protein
reservoir (91). In hindsight, many of the reports used inadequate
procedures that subsequently have been replaced by mass
spectrometry and other more sophisticated procedures.
For years, the mechanism of arsenic species having a +3
oxidation state has been claimed to be their reaction with thiol
compounds, e.g., GSH, cysteine, lipoic acid, and/or the thiols
of proteins. Very few of these proteins have been identified.
Such a broad description of arsenic binding and toxicity,
although correct, is no longer adequate with today’s highly
sophisticated proteomic techniques such as DIGE and mass
spectrometry.
Certainly, protein binding of arsenic species is implicated in
their metabolism and toxicity. The arsenic species with a +3
oxidation state are chemically more reactive than the +5 species
(92). The former species, however, each have a different degree
of reactivity. For example, arsenite has three binding sites,
MMA(III) has two, and DMA(III) has one. This has been
extensively discussed in an excellent review by Carter et al.
(93). The review, written mainly by Professor Carter, is highly
recommended reading for those who wish to learn about the
many facets and needs of arsenic toxicology research from a
chemical point of view.
7.1. Hemoglobin Binding
A collaborative effort has used chemical and biological
techniques to elucidate the binding of +3 arsenic species to rat
and human hemoglobin (93). In the past, the prevailing
speculation had been that the increased concentration of arsenic
in rat blood was due to DMA(V) binding to hemoglobin (93,
94). Because one of the major changes in understanding arsenic
metabolism and toxicity has been the result of the recent
evidence demonstrating the greater reactivity and toxicity of
MMA(III) and DMA(III) (44, 45, 92), the investigators studied
the affinity of these +3 arsenic species for rat or human
hemoglobin. They used chromatography and nanoelectrospray
mass spectrometry. The apparent binding constants (Table 4)
showed that arsenic binding to rat hemoglobin was 3-16-fold
greater than for binding to human hemoglobin.
Aposhian and Aposhian
Table 4. Apparent Binding Constants (nK) for Trivalent Arsenicals
Binding to rHB and hHb (93)
nK (M-1)
arsenic species
rHb
hHb
iAs (III)
MMA(III)
DMA(III)
PhAs(III)O
.0233 × 105
.469 × 105
2.22 × 105
5.35 × 105
.007 × 105
.050 × 105
.136 × 105
.775 × 105
The binding was consistent with the number of reactive
cysteine residues in the R- and β-chains of hemoglobin. There
are three cysteines, Cys 13, Cys 104, and Cys 111, in each
R-chain and two cysteines, Cys 93 and Cys 125, in each β-chain
of rat hemoglobin. The rat tetramer contains two R- and two
β-units, a total of 10 DMA(III) molecules can be bound to the
10 sulfhydryl groups of rat hemoglobin. Human hemoglobin
contains only one cysteine in each R-chain (Cys 104) and two
in each β-chain (Cys 93 and Cys 112). It would be expected
that six DMA(III) molecules would bind to human hemoglobin.
However, the investigators suggest that hydrophobicity may also
be a factor. It is unfortunate that they did not incorporate some
ROS studies into this excellent paper (93).
These studies were extended to in vivo rat experiments (93).
Young rats were fed a diet containing 100 mg DMA(V)/kg for
72 days after which they were euthanized and plasma and red
cells were collected. The arsenic in red cells was predominantly
protein bound. The arsenic concentration in the plasma was 7.3
( 1.0 µM and that in the red blood cells (RBCs) was 1101 (
130 µM. Therefore, the arsenic concentration of rat RBCs under
these experimental conditions was 150 times greater than that
found in the plasma.
Hayakawa et al. (5) incubated at pH 7.0 a 20% rat liver
homogenate for 20 min at 37 °C with 1 µM various arsenic
species. The reaction was stopped by boiling, centrifuging the
pellet, and acid digestion, and the arsenic concentration was
measured by ICP-MS. The binding of MMA(III) was the
greatest. Arsenite had 60% of the MMA(III) binding activity.
DMA(III), arsenate, MMA(V), and DMA(V) each had about
15%. Because the DMA(III) was produced by the reduction of
DMA(V) using metabisulfite-thiosulfate (5, 94), questions
remain about the interpretations of these results. The Reay-Asher
procedure not only would produce DMA(III) but also significant
amounts of thioarsenicals (95, 96).
7.2. Metallothionein Binding
Metallothionein is an unusual polypeptide. It contains 20
cysteine residues. It has been implicated in the detoxication of
many toxic metals (97), scavenging of free radicals (98), and
metal transport (99). There have been many unconvincing
attempts to connect it with arsenic metabolism. A straightforward, careful study of how arsenic compounds having a +3
oxidation state interact with apo-metallothioneins has appeared
(100) using commercially available rabbit metallothionein II.
Each metallothionein molecule bound up to six As(III), 10
MMA(III), and 20 DMA(III) molecules. Because arsenite has
three binding sites and metallothioneins have 20 cysteine
residues, the maximum number of arsenite binding to metallothionein should be six. MMA(III) has two binding sites so
up to 10 of this arsenic species would be expected to bind to
metallothionein. Because DMA(III) has only one binding site,
it would be expected that MT would bind up to 20 DMA(III).
Their experimental results confirmed the theoretical expectations, and no binding of TMAO(V) was detected. Neither could
complexes of MMA(V) nor DMA(V) with metallothionein be
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Chem. Res. Toxicol., Vol. 19, No. 1, 2006 11
Figure 9. Example of information obtainable by DIGE.
Figure 8. DIGE: Sample preparation.
detected. It would be of interest to do such experiments in vivo
to determine its relevance to arsenic modulations in the whole
animal. While this study is a revealing one, it would have been
improved if experiments or previously reported data were used
to estimate the relative affinities of these arsenic species as
compared to cadmium, mercury, and lead.
7.3. Other Pertinent Proteomic Papers
An excellent, innovative paper from van Houten’s laboratory
combining genomics and proteomics of metals and yeast is
unusual for its comprehensiveness, clarity, and relevance (101).
Kitchen and Wallace (102) have reported arsenite binding to
synthetic peptides based on the Zn finger region and estrogenbinding region of the human estrogen receptor. One must not
ignore older, classic papers on arsenic toxicology by Vahter.
For example, the first indication of some animals not methylating inorganic arsenic but detoxifying by protein binding was
presented by her (88, 89).
more specific knowledge as to the exact role of specific proteins
in arsenic intoxication and detoxication. Using two-dimensional
electrophoresis procedures in which protein extracts from two
subjects can be electrophoresed together by using two fluorescent dyes followed by MS of protein spots may quicken the
understanding of the role of proteins in arsenic metabolism. A
recent study clearly has shown that the differences in the number
of cysteine residues in human and mouse hemoglobin are
responsible for the greater accumulation of arsenic species in
rat blood.
8. Areas of Concern and Conclusions
In the authors’ laboratory, DIGE has been used to study the
liver proteins of mice given sodium arsenite in drinking water
each day for 14 days. Proteomics (DIGE) has many advantages
over microgene array analysis. The latter depends on measuring
the gene transcript (mRNA). The former measures protein
synthesis including any posttranslational modifications. Not
everything that is transcribed is translated. This proteomic
procedure (103) is becoming an important exploratory procedure
in toxicology research. It can compare the relative amounts of
specific proteins in body fluids or tissues of two subjects by
placing extracts on the same space of a single gel and performing
an electrophoresis first by isoelectric point and second by
molecular weight (Figure 8). By having a dye that fluoresces a
certain color in one extract and a dye of a different color in the
second extract, not only can the proteins be separated, but by
using a scanner and specific software, the relative amounts of
a specific protein in one sample can be determined to be more,
equal to, or less than in the other (Figure 9). The procedure is
expensive because of the costs of the dyes, use of a scanner
plus appropriate software, and finally mass spectrometry of each
protein spot.
There are areas of concern that are related to most of the
five questions that we have addressed. Investigators need to keep
them in mind while they do research and deal with the enigma
of arsenic toxicology. Dose-response is one such area. An
attempt to characterize a dose-response relationship between
arsenic concentrations in drinking water in West Bengal, India,
and arsenic-induced skin keratoses and hyperpigmentation has
appeared (104). The average latency for skin lesions was 23
years from the first exposure. The lowest peak arsenic ingested
by a confirmed case was 115 µg As/L. Although such values
are helpful signposts, they need to be viewed with caution when
attempts are made to extend such values to other areas of the
world where genes, nutrition, and other factors may be different,
even though the drinking water contains similar concentrations
of arsenic. The major cause for such concern would be genetics
more specifically polymorphisms of the arsenic biotransformation genes in different ethnic groups. Climate differences in other
parts of the world also can influence the volume of water
consumed and the resulting arsenic exposure. Arsenic antagonists such as selenium salts (28) being present in different
concentrations in different countries may influence threshold
values. The above-mentioned dose-response study, hopefully,
is preceding more extensive studies.
The results of a paper (105) studying arsenic exposure and
the intellectual function of Bangladesh children need to be of
concern to policy makers and to financial organizations that
support research on the arsenic catastrophe in Bangladesh and
in parts of India. Exposure from drinking water with high levels
of arsenic was associated with reduced intellectual function in
a dose-related manner in these children. This important, highly
relevant paper by the Graziano group is further proof for the
need of environmental justice for the children of the poor.
7.5. Conclusion Five
8.1. Tolerance
With new proteomic techniques available, such as DIGE,
research in arsenic toxicology now may be expanded to acquire
Ever since the arsenic eaters of Styria were studied by British
physicians (106, 107) during the mid-1800s, the question of
7.4. DIGE
12 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
the mechanisms for the development of tolerance to inorganic
arsenic by humans has been a challenge to many arsenic
investigators. After all of these years, the challenge has not been
answered successfully. Romach et al. (108) using chronic
arsenite-exposed rat liver epithelial cells found that the cells
accumulated 87% less As as compared to controls. The tolerance
seemed to be acquired without changes in GSH or metallothionein. More studies of these fascinating arsenic tolerance
phenomena are needed including whether there is inducibility
of the arsenic biotransforming enzymes in human and rabbit
cells.
8.2. Inappropriate Procedures for Synthesis
The Reay and Asher procedure (94) was designed to reduce
arsenate to arsenite using metabisulfate-thiosulfate. Many
investigators have used this procedure believing that they were
converting MMA(V) to MMA(III) and DMA(V) to DMA(III).
Some neglected, however, to confirm the structure of the product
and/or to remove unreacted starting materials and unknown
products of the reaction. The results of most of these published
experiments that used the Reay and Asher procedure to prepare
MMA(III) and DMA(III) as enzyme substrates or tissue culture
media constituents require, at the minimum, reevaluation and,
at the maximum, disbelief. Most of these now questionable
studies were performed before the Reay and Asher procedure
was shown to produce thioarsenicals (95, 96).
8.3. Inappropriate Procedures for Urine Collection
Since the initial finding of MMA(III) in human urine by
Aposhian et al. (79), there has been concern about how to collect
and store urine in order to minimize oxidation of MMA(III).
The Le group has been a major contributor in trying to solve
this problem (109, 110, 111). A recent study by Valenzuela et
al. (112) found that MMA(III) was 7.4% and DMA(III) 49%
of the total urinary arsenic. These are the highest levels ever
reported especially for DMA(III), and the authors deserve to
be complimented on obtaining such results due to quickly
freezing the urine in dry ice and analysis within 6 h after
collection. However, because most studies of arsenic-exposed
populations take place in countries without adequate analytical
laboratories, the need continues for easy, convenient methods
of collecting and storing urine samples that cannot be analyzed
within a period of 6 h because of transporting time. Because it
is generally accepted that the matrix affects the stability of all
urinary arsenic species, the Valenzuela et al. results also might
have been influenced by reducing substances in urine originating
in the diet. This, however, does not minimize the importance
of their results. Continued efforts are needed to stabilize arsenic
species in the urine.
8.4. Compound Identification
Another area of concern has been the use of only one property
of a compound to identify it. For example, the use of only
retention times after HPLC or Rf after paper or thin-layer
chromatography leaves a great deal to be desired as far as
identification of new intermediates and metabolites. Mass
spectrometry now is available at most research institutions and
needs to be used for initial or confirmatory identification of
chemical structures.
8.5. Inadequacy of the Rat as a Model for Arsenic in
Humans
It has been known for many years that DMA will bind rat
RBCs to a greater extent than red cells of other species. (The
Aposhian and Aposhian
reader may want to review question five of this review at this
point). The extrapolation of arsenic toxicokinetic and metabolic
studies from rat experiments can lead to erroneous conclusions
since many investigators have ignored these different properties
of rat RBCs. In addition, the urinary arsenic species, as percent
of total arsenic, of the rat are very different than the human
(113). On the basis of urine arsenic species, if an animal must
be used, it appears that the hamster and rabbit are the most
reasonable and desirable. Rat studies should be viewed with
caution and the result should not be extended to or used as a
model for the human. Having said this, it needs to be realized
that the rat may be one of the few arsenic carcinogenic models
available at present. The best model system for studying arsenic
toxicology and risk assessment in the human remains the human.
Human tissues are available for in vitro studies, and the human
body can be used in vivo for ethical and safe excretion and
epidemiology studies.
8.6. Summary
We have tried to address five questions dealing with five of
the arms of arsenic toxicology: biotransformation, ROS, polymorphism, treatment, and protein binding. The first question,
“What enzyme is responsible for the methylation of arsenic
species in the human?”, still needs further investigative effort
to obtain an answer. The dilemma continues. For CYT 19 to
be accepted as the methylation enzyme of humans, purification
of the protein and its activity from surgically removed human
tissue is required. This has not been accomplished for either
CYT 19 or the rabbit methyltransferase.
Second, the conventional belief that arsenite inhibits PDH
and perhaps other dithiol-containing enzymes by chelating the
thiol groups now needs to be expanded to include ROS. The
latter also can be generated as an inhibitory agent by arsenicals.
Third, a number of polymorphisms in human GST ωs, CYT
19, and PNP have been reported. Two studies have linked these
polymorphisms with changes in urinary arsenic species. There
has been a minimum of investigations dealing with both studies
of polymorphisms of human genes known to be involved in
arsenic metabolism and the determinations of all of the possible
urinary arsenic species, especially MMA(III) and DMA(III). In
fact, the genetics of arsenic toxicity is a barren field at present.
Fourth, DMSA and DMPS are effective in mobilizing the
excretion of arsenic from the human. DMPS seems to be more
consistently effective in the clinical improvement of individuals
chronically exposed to arsenic. With the millions of people now
known to be consuming toxic amounts of arsenic in their
drinking water or food, a large-scale clinical trial of arsenic
antidotes is needed and recommended so that when remediation
of arsenic exposure is finally accomplished, arsenic body
burdens of exposed humans can be decreased safely.
Fifth, with new proteomic techniques available, such as DIGE,
research in arsenic toxicology now may be expanded to acquire
more specific knowledge as to the exact role of specific proteins
in arsenic intoxication and detoxication. Using two-dimensional
electrophoresis procedures in which protein extracts from two
subjects can be electrophoresed together by using two fluorescent dyes followed by MS of protein spots may quicken the
understanding of the role of proteins in arsenic metabolism. A
recent study clearly has shown that the differences in the number
of cysteine residues in human and mouse hemoglobin are
responsible for the greater accumulation of arsenic species in
rat blood.
While a number of recent papers have been emphasized in
this review, the need for confirmation of their conclusions by
ReViews
Chem. Res. Toxicol., Vol. 19, No. 1, 2006 13
other investigators is needed. The importance of their results at
this time, however, should not be minimized. Rather, they should
stimulate investigators to reexamine and expand their thinking
and investigations and, hopefully, attract new investigators.
Acknowledgment. We are grateful to Dr. Robert A. Zakharyan and Dr. Uttam Chowdhury for reading and critically
reviewing this manuscript. This review was written while the
research in our laboratory was supported in part by Superfund
Basic Research Program NIEHS Grant ES-04940, the Southwest
Environmental Health Sciences Center Grant P30-Es-06694
from the National Institute of Environmental Health Sciences,
and the Wallace Research Foundation.
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