A Speciation Study focused on the Identification of
Proximate Toxic Arsenic Metabolites
Kazuo T. Suzuki
Graduate School of Pharmaceutical Sciences, Chiba University
Inage, Chiba 263-8522, JAPAN
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
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Chemical species of arsenic
taken by humans and excreted into urine
Ingested forms
Inorganic
Organic
arsenite
arsenate
arsenosugars
arsenocholine
arsenobetaines
Excreted forms (urinary metabolites)
dimethylated arsenic (DMA)
(dimethylarsinic acid (DMAV))
Most probable pattern in the toxification of
arsenic in humans
Ingested forms
Arsenite or
Arsenate
Urinary metabolites
DMA (DMAV)
Reduction and methylation reactions
in the metabolism of arsenic
OH
O
iAsV
As OH
OH
OH
OH
O
As OH
iAsIII
OH
DMAV
CH3
MMAIII
OH As CH 3
iAsV arsenate
MMAV
monomethylarsonic acid
DMAV dimethylarsinic acid
OH
O As CH3
CH3
OH As OH
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MMAV
OH
CH3 As CH 3
reduction
methylation
DMAIII
iAs III
arsenite
MMAIII
monomethylarsonous acid
DMAIII dimethylarsinous acid
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
Reduction and methylation reactions
in the metabolism of arsenic
Detoxification pathway
or
Toxification pathway?
Which arsenic metabolites do cause
the arsenic toxicity?
What are the proximate toxic
arsenic metabolites?
Tri- and pentavalent forms of arsenic
Trivalent
iAsIII
MMAIII
DMAIII
More reactive
Pentavalent
iAsV
MMA V
DMA V
less reactive
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
47
Possible reactions for trivalent forms of arsenic
AsIII
AsV
2e-
reactive oxygen species (ROS),
radicals
RSH
RS-As
(RS-As(CH 3)2, (RS) 2-AsCH3, (RS) 3-As, RS-As=O)
Separation and subsequent analytical procedures
for arsenic in the body
Soluble
Free
Pentavalent arsenic
A
(iAsV, MMAV, DMAV )
Trivalent arsenic
B
(iAsIII, MMAIII, DMAIII)
Conjugated
Trivalent arsenic – GSH
C
(iAsIII(GS)3 , CH3 AsIII (GS)2, (CH3 )2AsIII(GS))
Trivalent arsenic - soluble proteins
D
Non-soluble
Conjugated
Trivalent arsenic - non-soluble proteins
E
Separation and subsequent analytical procedures
for arsenic in the body
Soluble
Free
Conjugated
Non-soluble
Conjugated
Pentavalent arsenic
Trivalent arsenic
Trivalent arsenic – GSH
Trivalent arsenic - soluble proteins
Trivalent arsenic - non-soluble proteins
A
B
C
D
E
A, B: Free arsenic can be speciated simultaneously on an anion or
cation exchange column by HPLC-ICPMS.
C: Trivalent arsenic conjugated with GSH can be speciated
simultaneously on an anion exchange column by HPLC-ICPMS.
D: Trivalent arsenic conjugated with thiol groups of soluble proteins
can be characterized on a gel filtration column by HPLC-ICP MS.
However, the arsenic species and the corresponding protein
have to be identified separately.
E: Trivalent arsenic conjugated with non-soluble proteins is not
applicable to HPLC separation. The arsenic species can be
identified after separation to free arsenic.
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Arsenic Contamination in Groundwater - Technical and Policy Dimensions
Why rats are less toxic to arsenic than other mammals?
What is the significant difference in the metabolism of
arsenic in rats compared with other animals?
What are the difference in the toxicity and hence metabolism of
arsenic between sensitive and tolerant animal species?
Characteristics of arsenic metabolism in rats
Longer biological half time in rats than in humans.
Why arsenic is more toxic in humans despite of
its shorter biological half time?
Specific accumulation of arsenic in red blood cells in rats.
Why does arsenic accumulate in RBCs in rats?
The major urinary metabolite in rats and
humans is believed to be DMA(DMAV).
Is it true?
Speciation of arsenic in the bile of rats after
an intravenous injection of arsenite
on a cation exchange column by HPLC-ICP MS
( A)
X 1/6
MMAI I I
30 min
V
MMA
0
2
4
6
8
10
Retention time (min)
iAsV
( B)
30 min
DMA
MMA V
V
i) Authentic
iAsIII
iAsV
DMA V
60 min
AsB
90 min
MMA III
120 min
ii) Reduced
MMA
iAs III
DMAIII
150 min
iii) Reduced
DMA
180 min
210 min
240 min
iv) As(GS) 3
270 min
v) CH3As(GS)2
300 min
0
2
4
6
8
Retention time (min)
10
0
3
6
9
12
15
Retention time (min)
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
49
Speciation of arsenic in the bile of rats after
an intravenous injection of arsenite
on an anion exchange column by HPLC-ICP MS
( A)
C H3 As(GS)2
As(GS) 3
MMA V
0
2 4
6 8 1 0 12
( B)
30 min
i) Authentic
D M AV
M M AV
AsB
60 min
i A s III
iAsV
90 m i n
iAs III
MMA III
ii) Reduced
MMA
120 min
D M A III
150 m i n
iii) Reduced
DMA
180 min
As(GS) 3
210 m i n
240 min
iv) As(GS) 3
270 m i n
v) CH 3As(GS) 2
300 min
0
2
4
6
8
10
12
0
Retention time (min)
2
4
6
8
10
12
Retention time (min)
Proposed metabolic pathway for arsenic after
an intravenous injection of arsenite in rats
urine
iAsIII
DMAIII DMAV
MMA III
bloodstream
RBCs
DMAV
DMA III
DMAIII
iAsIII
MMA III
DMAIII
MMAIII
bile duct
DMAV
iAsIII(SG)3
MMA V
iAsIII
iAsIII
liver
intestine
MMA III(SG)2 iAsIII
iAsIII(SG)3
MMAIII(SG)2
MMAIII
main route
bile excretion
Speciation of arsenic in the urine of rats after
an intravenous injection of arsenite
iAs III
cation exchange
DMAV
MMA V
iAsV
AsB
0
50
3
6
9
12
Retention time (min)
15
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
Selective uptake of DMA III by red blood cells (RBCs)
chemical species difference
Reduction
(-)
Reduction
(+)
m/z 75
DMAV
3
6
9
Retention time (min)
12
As (µg/mL medium)
A)
0
animal species difference
DMAIII
25
B)
As ( µg/mL medium)
20
15
Plasma As (µg/mL)
10
DMA V
DMAIII
5
0
0
1
2
3
Incubation time (hr)
13.3 µg/mL
14
12
10
8
6
4
2
0
A) rat
13.3 µg/mL
B) hamster
13.3 µg/mL
14
12
10
8
6
4
2
0
13.4 µg/mL
DMAV
DMAIII
C) mouse
4
0
1
2
D) human
3
4 0
1
2
Incubation time (hr)
3
4
Co-authors
Y. Shiobara, Y. Ogra and K. T. Suzuki
Animal species difference in the uptake of dimethylarsinous acid (DMAIII )
by red blood cells.
Chem. Res. Toxicol. 14, 1446-1452 (2001).
B.K. Mandal, Yasumi. Ogra and K.T. Suzuki
Identification of dimethylarsinous and momomethylarsonous acids in human urine
of the arsenic affected areas in West Bengal, India.
Chem. Res. Toxicol. 14, 371-378 (2001).
K.T. Suzuki, T. Tomita, Y. Ogra and M. Ohmichi
Glutathione-conjugated arsenics in the potential
hepato-enteric circulation in rats
Chem. Res. Toxicol. 14, 1604-1611 (2001)
Arsenic Contamination in Groundwater - Technical and Policy Dimensions
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Arsenic Contamination in Groundwater - Technical and Policy Dimensions