DSpace at VNU: Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

Toxicology and Applied Pharmacology 242 (2010) 352–362

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic
metabolism in residents of the Red River Delta, Vietnam
Tetsuro Agusa a,b, Hisato Iwata b,⁎, Junko Fujihara a, Takashi Kunito c, Haruo Takeshita a, Tu Binh Minh d,
Pham Thi Kim Trang d, Pham Hung Viet d, Shinsuke Tanabe b
a

Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, Japan
Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan
d
Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Vietnam National University, T3 Building, 334 Nguyen Trai Street,
Thanh Xuan District, Hanoi, Vietnam
b
c

a r t i c l e

i n f o

Article history:
Received 14 September 2009
Revised 29 October 2009
Accepted 6 November 2009
Available online 13 November 2009

Keywords:
Arsenic
Glutathione S-transferase ω1 (GSTO1)
GST ω2 (GSTO2)
GST π1 (GSTP1)
GST μ1 (GSTM1)
GST θ1 (GSTT1)
Genetic polymorphism
Vietnam

a b s t r a c t
To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic
polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic
concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam.
Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and
Ala236Val, GST ω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1
(GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this
population. GSTO1 Glu155del hetero type showed higher urinary concentration of AsV than the wild homo
type. Higher percentage of DMAV in urine of GSTM1 wild type was observed compared with that of the null
type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in
this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic
arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from AsV to
AsIII. Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and
genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with
arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the
associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.
© 2009 Elsevier Inc. All rights reserved.

Introduction
Inorganic arsenic (IA) is known to be a carcinogenic chemical in

human. Arsenic contamination in groundwater is one of the most serious
health concerns in the world (Mandal and Suzuki, 2002; Nordstrom,
2002; Smedley and Kinniburgh, 2002), especially in developing countries
where several areas do not have a public water supply system as yet.
Since 2001, our research group has investigated arsenic pollution in the
groundwater and residents from Vietnam and Cambodia (Agusa et al.,
2002, 2004, 2005, 2006, 2007, 2009a, 2009b, 2009c; Iwata et al., 2007;
Kubota et al., 2006). We found high concentrations of arsenic (up to
1930 μg/l) in the groundwater exceeding 10 μg/l of WHO water standard
value (WHO, 2004) and suggested that people in the area may be
exposed to high levels of arsenic through the water consumption.
In human, IA ingested through the drinking water and food is
metabolized to dimethyl arsenic. Two pathways are hypothesized to
account for the metabolism of IA: a classical scheme consists of a
series of reductions and oxidations coupled with methylations
⁎ Corresponding author. Fax: +81 89 927 8172.
E-mail address: (H. Iwata).
0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2009.11.007

(Challenger, 1945; Cullen and Reimer, 1989) and a recently proposed
concept, the reductive methylation by interaction with binding
proteins (Hayakawa et al., 2005; Naranmandura et al., 2006). In the
biotransformation process, two enzymes, arsenic (+ 3 oxidation
state) methyltransferase (AS3MT) and glutathione S-transferase ω
(GSTO), are required in a variety of animals including human
(Aposhian and Aposhian, 2006). GST is a phase II enzyme that can
detoxify xenobiotics by catalyzing the conjugation with reduced
glutathione. GST superfamily includes seven classes, α, μ, ω, π, θ, σ,
and ζ, and the function of GSTO is different from other members of

GST superfamily (Board et al., 2000). Among GSTO isoforms, GSTO1 is
involved in the reduction activities of arsenate (AsV), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) (Zakharyan
and Aposhian, 1999; Zakharyan et al., 2001, 2005). GSTO2 which was
recently identified by Whitbread et al. (2003) could catalyze the
reduction of MMAV and DMAV, but its activity of DMAV reductase was
remarkably lower than that of GSTO1 (Schmuck et al., 2005).
It is anticipated that there is a large variation in susceptibility to toxic
effect by IA among individuals and ethnics, depending on the difference
in IA metabolism (Vahter, 2000). Polymorphism(s) in the genes that are
responsible for the metabolism of arsenic compounds may contribute to


T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

the variability in biotransformation of IA (Loffredo et al., 2003; Vahter,
2000, 2002). Our recent findings indicate significant associations of
genotypes and haplotypes in AS3MT, which catalyzes the methylation of
arsenite (AsIII) and monomethylarsonous acid (MMAIII) (Lin et al., 2002;
Wood et al., 2006), with arsenic methylation capacity estimated by
urinary arsenical profile in a Vietnamese population (Agusa et al., 2008,
2009b). Some researchers have reported the relevance of genetic
polymorphisms in GSTO1 and O2 to arsenic metabolism by in vitro
assays (Tanaka-Kagawa et al., 2003; Whitbread et al., 2003; Schmuck et
al., 2005) and in human studies (Marnell et al., 2003; Meza et al., 2005;
Paiva et al., 2008). Furthermore, there are some reports on significant
associations of single nucleotide polymorphisms (SNPs) in GST π
(GSTP1) and wild/null genotypes in GST μ (GSTM) and GST θ (GSTT)
with biotransformation of arsenic (Chiou et al., 1997; Kile et al., 2005;
Marcos et al., 2006; Zhong et al., 2006; Lin et al., 2007; McCarty et al.,
2007; Steinmaus et al., 2007). However, there is no information on the

distribution of gene polymorphisms in GSTs and their relation to the
arsenic metabolism in Vietnamese. In the present study, we investigated
whether genetic polymorphisms in the members of GST superfamily,
GSTO1, GSTO2, GSTM1, GSTP1, and GSTT1, can affect arsenic metabolism
in residents from the Red River Delta, Vietnam. The co-influence of
genetic polymorphisms in GSTs and other factors (sex, age, body mass
index (BMI), arsenic level in drinking water, and AS3MT genotypes) on
the accumulation and metabolism of arsenic was also examined.
Materials and methods
Samples. Detailed information on samples was presented in our
previous work (Agusa et al., 2009b). Well water (n = 28), human hair
(n = 99), urine (n = 100), and blood (n = 100) samples were
randomly collected in March 2006 from Hoa Hau (HH) and Liem

353

Thuan (LT) in Ha Nam Province, which is located in the Red River
Delta, Vietnam. The informed consent was obtained from all the
participants, and also this study was approved by the Ethical
Committee of Ehime University, Japan. Data on concentrations of
total arsenic in water and human hair, and arsenic compounds in
urine (Agusa et al., 2009b), and cumulative arsenic exposure level are
summarized in Table 1. Cumulative As exposure level (mg) was
estimated from the As level in groundwater (mg/L), year of tube-well
usage (year), annual ingestion rate of groundwater (182.5 days/year),
and daily water consumption (3 L/day). All samples were kept at −25
°C in a freezer of the Environmental Specimen Bank (es-BANK) in Ehime
University (Tanabe, 2006) until the following analyses.
Analyses of arsenic. The analytical method of arsenic was described
in more detail elsewhere (Agusa et al., 2009b). Total arsenic (TA) in

water and human hair samples was analyzed with an inductively
coupled plasma-mass spectrometer (ICP-MS; HP-4500, HewlettPackard, Avondale, PA, USA) using internal calibration method.
Arsenic compounds including arsenobetaine (AB), DMAV, MMAV,
AsIII, and AsV in urine sample were measured with a high performance
liquid chromatograph (HPLC; LC10A Series, Shimadzu, Kyoto, Japan)—
ICP-MS. To separate each arsenical, a polymer-based anion exchange
column (Shodex Asahipak ES-502N 7C) was used with 15 mM citric
acid (pH 2.0 with nitric acid) (Agusa et al., 2009b).
In the present study, sum of all arsenic compounds and inorganic
arsenic (AsIII + AsV) detected in urine sample are denoted as SA and
IA, respectively. Percentages of AB, AsIII, AsV, MMAV, DMAV and IA to
SA in human urine were denoted as %AB, %AsIII, %AsV, %MMAV, %DMAV
and %IA, respectively. Urinary creatinine was determined at SRL, Inc.
(Tokyo, Japan). Concentrations of arsenic compounds in urine were
expressed as μg As/g on a creatinine basis. Because it is considered

Table 1
Information on water and human samples from Hoa Hau and Liem Thuan in Vietnam.
Location
Groundwater
No.
Used period (years)a
Well depth (m)a
TA (μg/l)b
Filtered water
No.
TA (μg/l)b
Drinking watere
No.
TA (μg/l)b

Subjects
No.
No. of male/female
Age (years)a
Residential time (years)a
Height (cm)a
Weight (kg)a
No. of smoker/non-smoker
No. of drinker/non-drinker
BMIa
Cumulative arsenic exposure (mg)b
Hair TA (μg/g)b
Urinary SA (μg/g creatinine)b
Urinary AB (%)a
Urinary DMAV (%)a
Urinary MMAV (%)a
Urinary AsIII (%)a
Urinary AsV (%)a

Hoa Hau

Liem Thuan

p-value

15
9 (5.5–13)
14 (8–16)
368 (163–502, and 2120 (an outlier))

13
6 (1–16)
15 (12–24)
1.4 (0.7–6.8)

0.049c
N 0.05c
b 0.001c

10
18.9 (3.2–143)

9
2.0 (1.0–4.9)

b 0.001c

15
50.1 (3.2–486)

13
1.7 (0.9–4.9)

b 0.001c

51
22/29
37 (11–60)
33 (3–60)
156 (137–173)

48 (27–66)
14/37
14/37
20 (14–26)
306 (17.6–12800)
0.351 (0.028–2.94)
92.6 (45.2–365)
22.7 (4.0–56.8)
55.9 (32.6–77.2)
10.6 (2.9–17.8)
8.5 (0–20.3)
2.3 (0–11.1)

49
22/27
34 (11–70)
31 (6–65)
150 (121–169)
44 (22–67)
6/43
10/39
19 (12–29)
4.8 (1.7–13.4)
0.232 (0.068–0.690)
97.9 (38.6–397)
19.6 (3.1–58.6)
59.0 (29.1–78.9)
10.0 (4.8–20.9)
8.7 (0–19.8)
2.7 (0–11.3)

N 0.05d
N 0.05c
N 0.05c
0.002c
0.027c
N 0.05d
N 0.05d
N 0.05c
b 0.001c
b 0.001c
N 0.05c
N 0.05c
N 0.05c
N 0.05c
N 0.05c
N 0.05c

Abbreviations: TA, total arsenic; BMI, body mass index (weight (kg)/height (m)2); SA, sum of arsenic compounds; AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV,
monomethylarsonic acid; AsIII, arsenite; AsV, arsenate.
a
Arithmetic mean and range.
b
Geometric mean and range.
c
t-test.
d
χ2 test.
e
In a house equipped with sand filter, filtered water instead of raw groundwater is assumed to be consumed.

354
Table 2
Information on primer sequences, annealing temperatures, restriction enzymes, and fragment sizes of the amplified products and frequencies of allele and genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam.
Amino acid
position

rs numbera

Functional
context

Nucleotide
change

Amino acid
change

Primer sequences

Temp.
(°C)

Restriction
enzyme

PCR method

Fragment size (bp)

Allele
Genotype
frequency (%) frequency (%)

GSTO1

140

rs4925

exon 4

c→a

Ala→Asp

5′-GAACTTGATGCACCCTTGGT-3′
5′-TGATAGCTAGGAGAAATAATTAC-3′

60

Cac8I

PCR-RFLP

Ala: 0.900
Asp: 0.100

GSTO1

155

rs56204475 exon 4

agg→del

Glu→del

GSTO1

208

rs11509438 exon 6

g→a

Glu→Lys

5′-GCTAGGAGAAATAATTACCTCTAGC-3′ 60
5′-GAATTTACCAAGCTAGAGGAGGT-3′
5′-GACCAAGCCAGCATTTTAGG-3′
5′-GCAGGACAGCTTTCTGCTTT-3′
5′-GACCTAGCTCACACCTTTCAT-3′
37
5′-CAAAGCGCTTGGCTGTTGATGTC-3′

Ala/Ala: 68, 186
Ala/Asp: 68, 186, 254
Asp/Asp: 254

Glu/Glu: 200, 472
Glu/del: 200, 315, 472
del/del: 315, 472

Glu: 1.000
Lys: 0

GSTO1

217

rs15032

exon 6

c→a

Thr→Asn

5′-GACCTAGCTCACACCTTTCAT-3′
5′-CAAAGCGCTTGGCTGTTGATGTC-3′

GSTO1

236

rs11509439 exon 6

c→t

Ala→Val

GSTO2

142

rs156697

exon 5

a→g

GSTP1

105

rs1695

exon 5

a→g

Glu/Glu: 154, 266
Glu/Lys: 154, 266, 420
Lys/Lys: 420
Thr/Thr: 335
Thr/Asn: 114, 221, 335
Asn/Asn: 114, 221
Ala/Ala: 116, 192
Ala/Asp: 116, 192, 308

Asp/Asp: 308
Asn/Asn: 185
Asn/Asp: 63, 122, 185
Asp/Asp: 63, 122
Ile/Ile: 176
Ile/Val: 84, 92, 176
Val/Val: 84, 92
Wild: 210
Null: 0
Wild: 473
Null: 0

GSTM1
GSTT1
a

CTPP

MboII

PCR-RFLP

50

MseI

PCR-RFLP

5′-CTGTGATGTCATCCTAGTTG-3′
5′-CATGCAACCTGAACCTTGGT-3′

50

StuI

PCR-RFLP

Asn→Asp

5′-AGGCAGAACAGGAACTGGAA-3′
5′-GAGGGACCCCTTTTTGTACC-3′

63

MBoI

PCR-RFLP

Ile→Val

5′-ACCCCAGGGCTCTATGGGAA-3′
5′-TGAGGGCACAAGAAGCCCCT-3′

55

BsmAI

PCR-RFLP

5′-GAACTCCCTGAAAAGCTAAAGC-3′

5′-GTTGGGCTCAAATATACGGTGG-3′
5′-TTCCTTACTGGTCCTCACATCTC-3′
5′-TCACCGGATCATGGCCAGCA-3′

62

Rs numbers were cited from NCBI SNP Database ( />
62

Allele specific
multiplex PCR
Allele specific
multiplex PCR

Glu: 0.955
del: 0.045

Thr: 1.000
Asn: 0
Ala: 1.000
Asp: 0
Asn: 0.780
Asp: 0.220
Ile: 0.845
Val: 0.155

Ala/Ala: 0.810
Ala/Asp: 0.180
Asp/Asp: 0.010
Glu/Glu: 0.910

Glu/del: 0.090

Glu/Glu: 1.000
Glu/Lys: 0
Lys/Lys: 0
Thr/Thr: 1.000
Thr/Asn: 0
Asn/Asn: 0
Ala/Ala: 1.000
Ala/Asp: 0
Asp/Asp: 0
Asn/Asn: 0.610
Asn/Asp: 0.340
Asp/Asp: 0.050
Ile/Ile: 0.690
Ile/Val: 0.310
Val/Val: 0
Wild: 0.580
Null: 0.420
Wild: 0.700
Null: 0.300

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

Gene
symbol


T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

that AsV, IA, and MMAV are metabolized to AsIII, MMA, and DMA,
respectively, in the human body, concentration ratios of AsIII/AsV (III/
V), MMAV/IA (M/I), and DMAV/MMAV (D/M) in human urine are
used as metabolic index for each arsenical.
Genotyping of polymorphisms in GSTO1, GSTO2, GSTP1, GSTM1, and
GSTT1. Genomic DNA was extracted from the blood of 100 subjects
using a QIAamp DNA mini kit (Qiagen, Chatworth, CA). Reference
sequence of each GST was based on the DNA Data Bank of Japan (DDBJ);
accession numbers of GSTO1, GSTO2, GSTP1, GSTM1, and GSTT1 are
AY817669, AY191318, AY324387, BC024005, and AB057594, respectively. DNA was subjected to PCR amplification in 10 μl reaction mixture
containing GoTaq® Green Master Mix (Promega, Madison, WI, USA) and
individual primer pairs corresponding to each mutation of GSTO1
Thr217Asn (threonine to asparagine substitution at amino acid base
217), GSTO1 Ala236Val, GSTP1 Ile105Val, GSTM1 wild/null, and GSTT1
wild/null. For the detection of GSTO1 Glu155del and Glu208Lys, the
amplification was conducted using genomic DNA, PCR buffer solution
(15 mM Tris–HCl, 50 mM KCl, pH 8.0), 1.5 mM MgCl2, 0.5 μM of each
primer, 200 μM dNTP, and 1.25 U Taq polymerase (AmpliTaq Gold,
Applied Biosystems, CA, USA) (Fujihara et al., 2007). A PCR mixture
consisting of PCR buffer, 1.5 mM MgCl2, 0.4 μM of each primer, 250 μM
dNTP, and 1 U Takara EX Taq DNA polymerase (Takara, Kyoto, Japan)
was used for genotyping of GSTO1 Ala140Asp and GSTO2 Asn142Asp
(Takeshita et al., 2009). Genetic polymorphisms of GSTO1 Ala140Asp,
Glu208Lys, Thr217Asn, and Ala236Val, GSTO2 Asn142Asp, and GSTP1
Ile105Val were detected by PCR restriction fragment length polymorphism (PCR-RFLP) using restriction enzymes. Genetic polymorphisms in
GSTT1 and M1 (wild or null) were identified by allele specific multiplex
PCR including β-globin as a positive control (Sreeja et al., 2005). GSTO1
Glu155del was detected by applying the method of confronting two-

355

pair primers analysis (CTPP) (Fujihara et al., 2007). The PCR products,
which were treated with restriction enzyme or were not treated, were
separated in 8% polyacrylamide gel by electrophoresis (300 V, 15 min)
and were detected by silver staining. The genotyping was carried out in
duplicate. The representativeness of nucleotide sequences for each
genotype was confirmed with a Genetic Analyzer (model 310, Applied
Biosystems Foster, CA, USA). Information on primers, annealing temperature, restricted enzyme, and fragment size is presented in Table 2.
Statistical analyses. Commercial software including StatView (version 5.0, SAS® Institute, Cary, NC, USA), SPSS (version 12, SPSS,
Chicago, IL, USA), and EXCEL Toukei (Version 6.05, Esumi Co., Ltd.,
Tokyo, Japan) were used for statistical analyses. One half of the value
of the respective limits of detection were substituted for those values
below the limit of detection and used in statistical analysis. Normality
for distribution of all variables was checked by Kolmogorov–
Smirnov's one sample test. To adapt parametric analyses, data
which showed non-normal distribution was log-transformed. Student's t-test and Tukey–Kramer test were conducted to find differences
in arsenic levels and compositions in the hair and urine among allele
types and genotypes of GST superfamily. χ2 test was employed for
checking sample size distribution in each group category. To assess
the factors affecting arsenic levels and composition in the urine and
hair, and metabolic capacity of arsenic, a stepwise multiple regression
analysis was executed. Genetic polymorphisms in GST superfamily
and cumulative As exposure level as well as SNPs in AS3MT, age, sex,
BMI, drinking water arsenic level which showed significant
relationships with arsenic levels and compositions in our previous
study (Agusa et al., 2009b) were incorporated in the regression
analysis as independent variables. To apply the regression model,
nominal variables (sex and genotypes of GST superfamily and AS3MT)

Fig. 1. Frequencies of genotypes of GST superfamily in the Vietnamese and the HapMap populations ( NA means no available data. VN:

Vietnamese in this study, CHB (H): Han Chinese in Beijing, China, CHD (D): Chinese in Metropolitan Denver, Colorado, JPT (J): Japanese in Tokyo, Japan, GIH (G): Gujarati Indians in
Houston, Texas, MEX (M): Mexican ancestry in Los Angeles, California, CEU (C): Utah residents with Northern and Western European ancestry from the CEPH collection, TSI (T):
Toscans in Italy, ASW (A): African ancestry in Southwest USA, LWK (L): Luhya in Webuye, Kenya, MKK (K): Maasai in Kinyawa, Kenya, YRI (Y): Yoruba in Ibadan, Nigeria.


356

Table 3
Concentrations (geometric mean and range) of arsenic compounds in urine and total arsenic in hair for each genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam.

GSTO1 Ala140Asp
Ala/Ala
Ala/Asp
Asp/Asp
Ala/Asp +
Asp/Asp
GSTO1 Glu155del
Glu/Glu
Glu/del
GSTO2 Asn142Asp
Asn/Asn
Asn/Asp
Asp/Asp
Asn/Asp +
Asp/Asp
GSTP1 Ile105Val
Ile/Ile
Ile/Val
GSTM1
Wild

Null
GSTT1
Wild
Null

n

Urine

Hair
V

V

III

AB

DMA

81
18
1
19

15.2 (2.1–232)
20.6 (4.9–71.9)
18.9
20.5 (4.9–71.9)

52.6 (22.5–268)
57.0 (20.2–121)
46.5
56.4 (20.2–121)

9.3 (3.5–23.1)
9.0 (4.3–23.9)
11.1
9.1 (4.3–23.9)

7.0 (b 1.0–26.6)
6.7 (b 1.0–32.2)
11.6
6.9 (b 1.0–32.2)

91
9

16.1 (2.1–232)
16.0 (5.3–44.2)

53.4 (20.2–268)
52.3 (34.5–81.1)

9.3 (3.5–23.9)
8.7 (5.4–12.2)

61
34
5

39

15.8
17.3
12.3
16.6

51.3
56.7
55.6
56.6

(22.5–268)
(20.2–121)
(34.5–81.1)
(20.2–121)

69
31

18.1⁎ (2.9–232)
12.4⁎ (2.1–72.6)

58
42
70
30

IA

SA

TA

1.6 (b 1.0–19.1)
1.7 (b 1.0–12.8)
b 1.0
1.6 (b 1.0–12.8)

9.6 (3.1–35.0)
10.2 (4.5–38.2)
11.6
10.3 (4.5–38.2)

93.4 (38.6–397)
104 (40.1–227)
88.0
103 (40.1–227)

0.300 (0.028–2.94)
0.229 (0.099–0.468)
NA
0.229 (0.099–0.468)

7.1 (b 1.0–32.2)
6.3 (4.0–10.0)

1.7⁎ (b1.0–19.1)
0.7⁎ (b1.0–3.0)

10.1⁎ (3.1–38.2)
6.8⁎ (4.0–10.0)

96.0 (38.6–397)
86.8 (49.5–129)

0.289 (0.028–2.94)
0.256 (0.129–0.526)

8.7 (3.5–23.1)
10.4 (4.3–23.9)
8.9 (5.4–11.8)
10.2 (4.3–23.9)

7.0
7.1
6.5
7.0

(1.7–26.6)
(b 1.0–32.2)
(4.2–10.0)
(b 1.0–32.2)

1.5 (0.5–19.1)
2.1 (b 1.0–12.8)
b 1.0 (b1.0)
1.7 (b 1.0–12.8)

9.3 (3.1–35)

11.2 (4.0–38.2)
6.5 (4.2–10.0)
10.4 (4.0–38.2)

91.6 (38.6–397)
103 (57.8–227)
87.0 (49.5–117)
101 (49.5–227)

0.296
0.265
0.311
0.271

55.3 (20.2–268)
49.1 (22.5–85.9)

9.9⁎ (3.8–23.9)
8.0⁎ (3.5–17.7)

8.3⁎⁎⁎ (b 1.0–32.2)
4.7⁎⁎⁎ (b 1.0–16.3)

1.7 (b 1.0–19.1)
1.3 (b 1.0–12.8)

11.1⁎⁎⁎ (3.2–38.2)
7.2⁎⁎⁎ (3.1–21.3)

102⁎ (38.6–397)

81.9⁎ (40.1–166)

0.294 (0.028–2.94)
0.268 (0.128–0.691)

13.6⁎ (2.1–72.6)
20.2⁎ (3.8–232)

55.9 (22.5–268)
50.0 (20.2–132)

9.2 (3.5–23.9)
9.4 (3.8–23.1)

7.1 (1.1–32.2)
6.9 (b 1.0–26.6)

1.6 (b 1.0–19.1)
1.5 (b 1.0–11.7)

9.8 (3.1–38.2)
9.6 (4.1–28.6)

95.0 (45.2–365)
95.3 (38.6–397)

0.314 (0.068–2.94)
0.251 (0.028–0.691)

15.5 (2.1–232)

17.5 (2.9–78.4)

54.5 (22.5–268)
50.8 (20.2–117)

9.2 (3.5–23.9)
9.6 (4.3–21.6)

7.0 (b 1.0–32.2)
7.1 (b 1.0–16.3)

1.6 (b 1.0–19.1)
1.6 (b 1.0–13)

9.7 (3.1–38.2)
9.8 (4.5–27.7)

95.3 (38.6–397)
94.8 (55.2–225)

0.271 (0.028–2.94)
0.324 (0.099–2.67)

(2.1–232)
(2.9–71.9)
(5.3–32.7)
(2.9–71.9)

MMA

As

As

V

(0.028–2.94)
(0.091–2.67)
(0.134–0.526)
(0.091–2.67)

Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); SA, sum of arsenic compounds; TA, total arsenic; NA, not available.
⁎ p b 0.05.
⁎⁎⁎ p b 0.001.

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

Gene and
genotype


Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); III/V, AsIII/AsV; M/I, MMAV/IA; D/M, DMAV/MMAV; NC, not calculated.
⁎ p b 0.05.
⁎⁎ p b 0.01.

6.4 (2.0–13.1)
5.6 (3.2–10.8)
1.0 (0.4–2.6)
1.0 (0.4–2.5)
20.2 (3.1–58.6)

23.4 (4.0–51.0)
70
30

58.5 (29.1–78.9)
54.8 (34.7–77.2)

10.2 (2.9–20.9)
10.6 (5.3–17.7)

8.6 (0–20.3)
8.6 (0–16.2)

2.5 (0–11.3)
2.5 (0–9.9)

11.1 (4.1–23.5)
11.2 (4.1–21.8)

3.6 (0.1–13.3)
3.4 (0.4–11.3)

6.5 (2.4–13.1)
5.6 (2.0–10.8)
1.0 (0.4–2.6)
1.1 (0.4–2.5)
18.5⁎ (3.1–56.8)
24.9⁎ (5.6–58.6)
58
42

60.1⁎⁎ (32.6–78.9)
53.7⁎⁎ (29.1–77.2)

10.3 (2.9–20.9)
10.3 (5.1–17.8)

8.7 (0.6–20.3)
8.6 (0–17.8)

2.5 (0–11.3)
2.4 (0–11.1)

11.2 (4.1–23.5)
11.0 (4.1–23.3)

3.4 (0.1–9.5)
3.7 (0.3–13.3)

6.0 (2.0–13.1)
6.5 (3.2–11.5)
0.9⁎⁎ (0.4–2.5)
1.2⁎⁎ (0.4–2.6)
2.5 (0–11.3)
2.4 (0–10.1)
22.2 (3.2–58.6)
18.8 (3.1–56.8)
69
31

55.7⁎ (29.1–78.9)
61.2⁎ (32.6–77.2)

10.3 (4.8–20.9)
10.3 (2.9–16.8)

9.3⁎ (0 –20.3)
7.2⁎ (0–17.8)

11.8⁎ (4.2–23.5)
9.6⁎ (4.1–23.2)

4.0⁎ (0.2–13.3)
1.9⁎ (0.1–6.1)

6.3 (2.0–13.1)
5.8 (2.4–11.1)
6.3 (5.0–6.9)
5.9 (2.4–11.1)
1.0 (0.4–2.6)
1.0 (0.4–1.9)
1.5 (1.0–2.5)
1.0 (0.4–2.5)
2.4 (0–11.3)
3.0 (0–9.9)
0 (0–2.4)
2.6 (0–9.9)

GSTO1 Ala140Asp
Ala/Ala

Ala/Asp
Asp/Asp
Ala/Asp + Asp/Asp
GSTO1 Glu155del
Glu/Glu
Glu/del
GSTO2 Asn142Asp
Asn/Asn
Asn/Asp
Asp/Asp
Asn/Asp + Asp/Asp
GSTP1 Ile105Val
Ile/Ile
Ile/Val
GSTM1
Wild
Null
GSTT1
Wild
Null

(3.1–58.6)
(4.0–47.3)
(9.0–39.4)
(4.0–47.3)
21.4
21.5
16.9
20.9
61

34
5
39

57.6 (29.1–78.9)
56.0 (34.9–77.4)
65.0 (43.4–72.7)
57.2 (34.9–77.4)

10.1
10.7
10.3
10.6

(2.9–17.8)
(4.8–20.9)
(8.6–11.3)
(4.8–20.9)

8.5
8.9
7.8
8.8

(2.4–20.3)
(0–19.8)
(4.1–10.2)
(0–19.8)

11 (4.1–23.0)

11.9 (4.1–23.5)
7.8 (4.1–10.2)
11.3 (4.1–23.5)

3.3 (0.2–13.3)
3.9 (0.1–11.3)
NC
3.9 (0.1–11.3)

6.2 (2.0–13.1)
6.1 (4.6–8.8)
1.0⁎ (0.4–2.6)
1.3⁎ (0.9–2.5)
21.3 (3.1–58.6)
20.6 (9.0–39.4)
91
9

57.0 (29.1–78.9)
61.1 (43.4–69.8)

10.3 (2.9–20.9)
10.1 (7.7–11.7)

8.7 (0–20.3)
7.6 (4.1–10.2)

2.7 (0–11.3)
0.7 (0–3.2)

11.4⁎ (4.1–23.5)
8.3⁎ (4.1–11.8)

3.6 (0.1–13.3)
2.4 (1.9, 2.9)

6.1 (2.0–13.1)
6.7 (3.5–11.1)
4.2
6.5 (3.5–11.1)
1.1 (0.4–2.6)
0.9 (0.5–1.9)
1.0
0.9 (0.5–1.9)
20.5 (3.1–58.6)
24.2 (4.8–47.3)
21.5
24.0 (4.8–47.3)
81
18
1
19

57.8 (29.1–78.9)
56.1 (34.9–77..4)
52.8
55.9 (34.9–77.4)

10.5 (2.9–20.9)
9.2 (4.8–14.8)

12.6
9.4 (4.8–14.8)

8.6 (0–20.3)
8.5 (0–15.2)
13.1
8.7 (0–15.2)

2.6 (0–11.3)
2.1 (0–7.7)
0
2.0 (0–7.7)

11.2 (4.1–23.5)
10.6 (4.1–17.9)
13.1
10.7 (4.1–17.9)

3.2 (0.2–13.3)
4.8 (0.1–11.3)
NC
4.8 (0.1–11.3)

D/M
M/I
III/V
%IA
%AsV
%AsIII
%MMAV

%DMAV
%AB
n
Gene and genotype

Table 4
Composition (arithmetic mean and range) of As compounds and concentration ratios of MMA/IAs and DMA/MMA in urine for each genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam.

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

357

were transformed to dummy variables. The multicollinearity of
independent variables was assessed by calculating the variance
inflation factor (VIF). Linkage disequilibrium and haplotype of SNPs
in GSTO1 were assessed by Haploview (version 4.0, Day Lab at the
Broad Institute Cambridge, MA, USA). P b 0.05 was considered to
indicate statistical significance.
Results and discussion
Distribution of genetic polymorphisms in GST superfamily
Allele and genotype frequencies for each gene are shown in
Table 2. There were no mutation alleles for GSTO1 Glu208Lys,
Thr217Asn, and Ala236Val in this population and thus the data on
these mutations were not included for further analysis. No mutation
homozygotes were found for GSTO1 Glu155del and GSTP1 Ile105Val.
All genotypes of GSTOs and GSTP1 Ile105Val followed the Hardy–
Weinberg Principle (p N 0.05). Among SNPs in GSTO1, there was no
significant linkage disequilibrium and haplotype.
Genotype frequencies for GST superfamily in this Vietnamese and
other populations which are available on HapMap (http://www.

hapmap.org/index.html.ja) are compared (Fig. 1). Proportions of
GSTO1 Ala140Asp, GSTO1 Glu208Lys, GSTO2 Asn142Asp, and GSTP1
Ile105Val genotypes in the Vietnamese population were similar to
those in Asian populations such as CHB (H) (Han Chinese in Beijing,
China groups), CHD (D) (Chinese in Metropolitan Denver, Colorado),
and JPT (J) (Japanese in Tokyo, Japan). However, even among the
Asian populations, frequencies of I/I and I/V genotypes for GSTP1
Ile105Val in the Vietnamese and Chinese (CHB (H) and CHD (D))
were largely different from those in the Japanese (JPT (J)). In addition,
although mutant homo types of GSTO1 Glu208Lys and GSTP1
Ile105Val were reported in other populations, no such substitution
was detected in the Vietnamese. There was no mutation in GSTO1
Ala236Val in the Vietnamese. Similarly, low mutation frequencies
were reported in other populations except for the Mexican. Genotype
frequencies of GSTO1 Ala140Asp, Glu155del, and Glu208Lys, and
GSTO2 Asn142Asp in the Japanese and Mongolian that have been
reported in our previous studies (Fujihara et al., 2007; Takeshita
et al., 2009) were close to the results on Vietnamese in this study
(Table 2).
For GSTO1 Glu155del and Thr217Asn, GSTM1 wild/null, and GSTT1
wild/null, the genotype frequencies of the present study were
compared with those in previous studies. Ninety-one percent of the
Vietnamese analyzed in the present study was the wild type of GSTO1
(Table 2) and the frequency was in the range (91–100%) of previous
studies (Whitbread et al., 2003; Fujihara et al., 2007; Paiva et al.,
2008).
No mutation allele was detected for GSTO1 Thr217Asn in the
present study population (Table 2). Up to date, there is no available
information on GSTO1 Thr217Asn mutation in any population,
although this type has been registered in NCBI SNP Database as

rs11509438. Tanaka-Kagawa et al. (2003) reported that GSTO1
Thr217Asn variant had lower MMAV reductase activity compared
with the wild type using in vitro assay, although the relevance of this
variant in arsenic metabolism is still unclear (Schmuck et al., 2005).
Null type frequencies of GSTM1 and T1 in Vietnamese were 42%
and 30%, respectively (Table 2). In the review article by Mo et al.
(2009), the prevalence rates were shown as 41.7–55.5% in Asians,
13.1–54.5% in Caucasians, 46.7% in American-Africans, and 26.9% in
Africans for GSTM1 null type and 41.9–52% in Asians, 11.1–28.6% in
Caucasians, 26.7% in American-Africans, and 36.6% in Africans for
GSTT1 null type. Compared with the results in the Asian populations,
frequency of GSTT1 deletion in the Vietnamese was lower, while
proportion of GSTM1 null type was within the range. On the contrary,
GSTT1 null type frequency in Taiwanese was 26% (Chiou et al., 1997),
which was similar to that in the Vietnamese. Lin et al. (2007) found


358
Table 5
Stepwise multiple regression of arsenic concentrations and compositions in urine and hair against sex, age, BMI, TA in drinking water, and polymorphisms in GST superfamily and AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam.
R2adj

p

Independent variable

β

P

Dependent variable

R2adj

p

Independent variable

β

p

%AB in urine

0.240

b 0.001

AS3MT g37853a (0 = others, 1 = a/a)
AS3MT t4740c (0 = others, 1 = t/t)
Sex (0 = female, 1 = male)

0.359
−0.235
−0.203

b 0.001
0.013
0.024

log AB in urine

0.189

b 0.001

AS3MT g37853a (0 = others, 1 = a/a)
AS3MT t35587c (0 = others, 1 = t/c)
GSTP1 Ile105Val (0 = Ile / Ile, 1 = Ile/Val)

0.355
−0.244
−0.217

b 0.001
0.009
0.019

%DMAV in urine

0.260

b 0.001

AS3MT t4740c (0 = others, 1 = t/t)
AS3MT g37853a (0 = others, 1 = a/a)
GSTM1 (0 = wild, 1 = null)
GSTP1 Ile105Val (0 = Ile / Ile, 1 = Ile/Val)
Age

0.282
−0.232
−0.223
0.204
0.182

0.003
0.015
0.017
0.022
0.044

log DMAV in urine

0.305

b 0.001

BMI
Age
AS3MT g35991a (0 = others, 1 = g/a)
AS3MT t4740c (0 = others, 1 = t/t)

−0.491
0.373
0.278
0.276

b 0.001
b 0.001

0.002
0.002

%MMAV in urine

0.306

b 0.001

Sex (0 = female, 1 = male)
AS3MT g12390c (0 = others, 1 = g/g)
AS3MT t5913c (0 = others, 1 = t/t)
AS3MT a6144t (0 = others, 1 = a/a)
AS3MT g37853a (0 = others, 1 = a/a)

0.294
0.676
−0.254
−0.512
−0.208

b 0.001
0.002
0.004
0.020
0.022

log MMAV in urine

0.165

b 0.001

AS3MT t35587c (0 = others, 1 = t/t)
AS3MT t4740c (0 = others, 1 = t/c)
Sex (0 = female, 1 = male)
GSTP1 Ile105Val (0 = Ile /Ile, 1 = Ile/Val)

0.248
−0.248
0.213
−0.204

0.009
0.009
0.023
0.031

%AsIII in urine

0.126

b 0.001

Sex (0 = female, 1 = male)
GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

0.307
−0.225

0.002
0.019

log AsIII in urine

0.183

b 0.001

GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)
Sex (0 = female, 1 = male)
AS3MT t4740c (0 = others, 1 = t/t)

−0.342
0.245
0.193

b 0.001
0.009
0.039

%AsV in urine

0.103

0.002

AS3MT g7395a (0 = others, 1 = g/a)
GSTO1 Glu155del (0 = Glu/Glu, 1 = Glu/del)

−0.299
−0.257

0.003
0.010

log AsV in urine

0.175

b 0.001

GSTO1 Glu155del (0 = Glu/Glu, 1 = Glu/del)
BMI
AS3MT g7395a (0 = others, 1 = g/a)
AS3MT t5913c (0 = others, 1 = t/t)

−0.286
−0.241
−0.237
0.186

0.004
0.012
0.015
0.050

%IA in urine

0.198

b 0.001

Sex (0 = female, 1 = male)
GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

0.408
−0.224

b 0.001
0.015

log IA in urine

0.291

b 0.001

GSTP1 Ile105Val (0 = Ile /Ile, 1 = Ile/Val)
Sex (0 = female, 1 = male)
AS3MT t4740c (0 = others, 1 = t/c)
BMI

−0.329
0.274
−0.241
−0.177

b 0.001
0.002

0.008
0.049

III/V in urine

0.186

0.001

AS3MT c14215t (0 = others, 1 = c/c)
GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

−0.362
−0.333

0.004
0.008

log SA in urine

0.198

b 0.001

M/I in urine

0.344

b 0.001

AS3MT g35991a (0 = others, 1 = a/a)
GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)
AS3MT t5913c (0 = others, 1 = t/t)
AS3MT t14558c (0 = t/t, 1 = t/c)
Sex (0 = female, 1 = male)
Age
AS3MT t4740c (0 = others, 1 = t/t)

−0.319
0.273
−0.279
0.224
−0.201
0.204
−0.205

0.001
0.002
0.002
0.013
0.017
0.020
0.022

BMI
GSTP1 Ile105Val (0 = Ile /Ile, 1 = Ile/Val)
Age
AS3MT t5913c (0 = others, 1 = t/t)

−0.456

−0.229
0.271
0.216

b 0.001
0.013
0.015
0.020

log TA in hair

0.282

b 0.001

BMI
log TA in drinking water
Age
Sex (0 = female, 1 = male)

−0.367
0.353
0.313
0.200

b 0.001
b 0.001
0.003
0.024

AS3MT g12390c (0 = others, 1 = g/g)
Sex (0 = female, 1 = male)
GSTM1 (0 = wild, 1 = null)
AS3MT t5913c (0 = others, 1 = t/t)

−0.301
−0.278
−0.271
0.240

b 0.001
0.002
0.003
0.008

D/M in urine

0.243

b 0.001

Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); III/V, AsIII/AsV; M/I, MMAV/IA; D/M, DMAV/MMAV; SA, sum of arsenic
compounds; TA, total arsenic; BMI, body mass index (weight (kg)/height (m)2).

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

Dependent variable


T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

that prevalence of GSTM1 and T1 deletion type were 71% and 35%,
respectively, in Hmong in China, and 42% and 47%, respectively, in Han
in China. Therefore, there may be large variations in the frequencies of
GSTM1 and T1 null type even in the Asian populations.
Potential effects of genetic polymorphisms in GST superfamily on arsenic
concentration and metabolism in Vietnamese
Since the hair can be a good indicator of chronic arsenic exposure
status, while arsenic level and speciation in the urine can represent
recent exposure and metabolism of arsenic in humans, respectively,
we measured arsenic in the hair and urine to examine their
relationships to genetic polymorphisms in GST isoforms. Although
arsenic concentration in drinking water as well as cumulative arsenic
exposure level showed significant regional difference (Table 1,
p b 0.001), arsenic level and compositional profile in the urine of
local people were not significantly different between HH and LT
(Table 1, p N 0.05) (Agusa et al., 2009b). Also, there were no significant
relationships between arsenic metabolism and TA in drinking water
or cumulative arsenic exposure (p N 0.05). Thus, the data of all donors
were pooled for analysis of the relationship between arsenic and
genotypes of GSTs. Concentrations and compositions of urinary
arsenicals and metabolic index for each genotype of GST superfamily
are shown in Tables 3 and 4. Because the sample sizes of the mutation
homo types of GSTO1 Ala140Asp (n = 1) and GSTO2 Asn142Asp
(n = 5) were small for the statistical analysis, these mutations were
included in hetero + homo type group for the further discussion.
Concentrations of AsV (p = 0.018) and IA (p = 0.050) in the urine of
subjects with GSTO1 Glu155del hetero type were significantly lower
than those with the wild type (Table 3). Furthermore, urinary %IAs was
also low in hetero type of GSTO1 Glu155del (p = 0.039, Table 4). On the

other hand, higher M/I value of GSTO1 Glu155del heterozygote was
observed when compared with the wild type (p = 0.039, Table 4).
Whitbread et al. (2003) found significantly higher activities of thioltransferase and GSH conjugation in mutation of GSTO1 Glu155del than
the wild type in an in vitro study. Hence, it might be suggested that if
methylation of IA by AS3MT occurs in the form of arsenic-glutathione as
proposed by Hayakawa et al. (2005), increased GSH conjugation by
GSTO1 Glu155del mutation protein enhances the methylation of IAs.
However, since the difference in M/I value between them was not so
large in the present study and also the association between GSTO1
Glu155del and first methylation index was not obvious in the multiple
regression analysis as shown later (Table 5), further studies are needed
to confirm the association. Schmuck et al. (2005) reported that GSTO1
Glu155del protein expressed in Escherichia coli exhibited higher MMAV
and DMAV reductase activities than the wild type. Because MMAIII and
DMAIII were not analyzed in the urine sample of the present study,
relationship between genetic polymorphisms in GSTO1 and methylated
trivalent arsenicals remains unclear for the subjects. In the case of
human studies, there was no relationship between GSTO1 Glu155del
and urinary arsenical in Mexican (Meza et al., 2005) and Chilean (Paiva
et al., 2008) in arsenic-contaminated regions. However, Marnell et al.
(2003) reported that only two donors with GSTO1 Glu155del substitution in Mexicans (n = 75) who were exposed to arsenic through the
consumption of drinking water showed unusual urinary arsenic profile;
one who has GSTO1 Glu155del and Glu208Lys substitutions had high %
AsV and low %DMAV in the urine and another one who has GSTO1
Ala140Asp substitution in addition to these two substitutions displayed
high %AsIII and low %DMAV in the urine compared to the rest of the study
population. In the present study, no such haplotype was found.
As for GSTM1, concentration (p = 0.018, Table 3) and composition
(p = 0.028, Table 4) of AB in the null type were significantly greater
than those in the wild type, although the reason was not clear.

Because AB can be less metabolized in human (Cullen and Reimer,
1989), the relationship for %AB might be partially due to a negative
correlation between %AB and %DMAV (p b 0.001). Urinary %DMAV

359

(p = 0.009) was low in GSTM1 null compared with that in the wild
type (Table 4), implying that GSTM1 null may affect %DMAV in the
Vietnamese. However, the result was not consistent with those in
previous studies. Chiou et al. (1997) reported slightly increased
urinary %IA for the null genotype of GSTM1 in Taiwanese. In the
workers occupationally exposed to arsenic in Chile (Marcos et al.,
2006) and women from arsenic-contaminated region in Argentine
(Steinmaus et al., 2007), GSTM1 null type had higher %MMAV than the
wild type. McCarty et al. (2007) found no significant association
between GSTM1 genotype and methylation ratios in Bangladesh
people with skin lesions. No relation of GSTM1 genotype to arsenic
level in toenail was reported in subjects from arsenic-endemic region
in Bangladesh (Kile et al., 2005), whereas arsenic concentrations in
urine and hair were high in GSTM1 null carriers exposed to indoor
combustion of high arsenic coal in China (Lin et al., 2007).
We found that GSTP1 Ile105Val homozygote had higher concentrations of AB, MMAV, AsIII, IA and SA, and %AsIII and %IA in the urine
than the heterozygote, whereas the opposite trend was observed for %
DMA (p b 0.05, Tables 3 and 4). Metabolic index of M/I in GSTP1
Ile105Val hetero type was significantly higher than that in the wild
type (p = 0.002, Table 4). Urinary III/V in the heterozygote of GSTP1
Ile105Val was about half of that in the wild homozygote (p = 0.027,
Table 4), suggesting that mutation of GSTP1 Ile105Val may lead to its
lower AsV reductase activity. Interestingly, activity of GST in
erythrocyte of GSTP1 Ile105Val wild type was higher than that of

the mutation type in the healthy Chinese, but AsV reduction activity
was not assessed (Zhong et al., 2006). Although GSTP1 Ile105Val
variant homo type showed a slightly (p = 0.085) higher %DMAV in the
urine than the wild type in Chileans (Marcos et al., 2006), the GSTP1
Ile105Val variant homo type was not observed in the population of
the present study.
There were no significant associations of GSTO1 Ala140Asp, GSTO2
Asn142Asp, and GSTT1 wild/null with concentrations and compositions of arsenic in the urine and hair (p N 0.05, Tables 3 and 4). The
result for the polymorphism of GSTO1 Ala140Asp was consistent with
the results reported in several in vitro studies; activities of MMAV
reductase and DMAV reductase of GSTO1 Ala140Asp mutation were
comparable to those of the wild type (Tanaka-Kagawa et al., 2003;
Schmuck et al., 2005), and no significant variation was detected in the
activities of thioltransferase and GST conjugation between GSTO1
Ala140Asp wild and mutation types (Whitbread et al., 2003).
According to the study of Mukherjee et al. (2006), protein expression
level of GSTO1 Ala140Asp mutation was similar to that of the wild
type, although the activity was not measured. In human studies, there
was no association of GSTO1 Ala140Asp with arsenic metabolism in
the Mexican (Meza et al., 2005), Hungarian, Romanian, and Slovakian
(Lindberg et al., 2007), and Chilean (Paiva et al., 2008) populations.
Like GSTO1, GSTO2 has six exons that are separated within 7.5 kb
nucleotide length on chromosome 10q24.3. The homology of amino
acid sequences between GSTO1 and GSTO2 is 64% (Whitbread et al.,
2003). Schmuck et al. (2005) reported that GSTO2 has a similar
capacity to reduce MMAV but a much lower activity for DMAV as
compared to GSTO1. Furthermore, GSTO2 Asn142Asp polymorphism
does not affect those reductase activities. This result may support our
results on GSTO2 Asn142Asp which showed no association with
arsenic metabolism (Tables 3 and 4).

Consistent with the results in Chilean (Marcos et al., 2006),
Argentina (Steinmaus et al., 2007), and Chinese (Lin et al., 2007)
subjects, GSTT1 wild/null polymorphism had no relevance to urinary
arsenic in Vietnamese of the present study (Tables 3 and 4). However,
there are some available data on significant relationships between
arsenic concentration and metabolism and the polymorphism. Chiou
et al. (1997) reported that in the residents exposed to arsenic through
drinking water, %DMAV in urine of subjects with GSTT1 null type was
higher than that in the wild type. Also, the interaction between GSTT1
wild type and secondary methylation ratio might increase the risk of


360

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

skin lesions among arsenic-exposed individuals in Bangladesh
(McCarty et al., 2007). Kile et al. (2005) reported higher concentration
of As in the nail of GSTT1 null type carriers in Bangladesh.
Concentration of TA in human hair showed a significant regional
difference (Table 1, p b 0.001) and was positively correlated with TA
level in drinking water (p b 0.001) (Agusa et al., 2009b) and
cumulative arsenic exposure level (p b 0.001). Therefore, the association of hair TA level with polymorphisms in GST superfamily was
assessed by ANCOVA, having correction with concentration of TA in
drinking water and cumulative arsenic exposure as covariates, but no
significant results were found (p N 0.05, Table 3). Similar to our results,
there were no significant associations between arsenic contents in
human hair and toenail and polymorphisms of GSTM1 and T1 in the
arsenic-contaminated region in Taiwan (Chiou et al., 1997). In
contrast, Lin et al. (2007) found high concentrations of arsenic in

both hair and urine of GSTM1 null carriers who were exposed to
arsenic from indoor coal combustion in Southwest Guizhou, China,
while there was no significant association between GSTT1 wild/null
and arsenic levels in hair and urine.
Potential effects of genetic polymorphisms in GST superfamily and AS3MT,
sex, age, BMI, arsenic level in drinking water, and cumulative arsenic
exposure level on arsenic concentration and metabolism in Vietnamese
In the previous study (Agusa et al., 2009b), we investigated the
influence of various factors on arsenical concentration and composition in the urine and hair in this Vietnamese population, including age,
sex, BMI, occupation, residential years, alcohol and smoking habits,
and TA level in drinking water, and 13 SNPs in AS3MT such as a4602g
(a to g substitution at nucleotide base 4602), t4740c, t5913c, a6144t,
g7395a, t8979a, g12390c, t12590c, c14215t, t14458c, t35587c,
g35991a, and g37853a, and revealed significant effects of sex, age,
BMI, concentration of TA in drinking water, and several SNPs in
AS3MT. Therefore, those factors might co-affect the results on
relationships between arsenic and GST genotypes examined in this
study. In the previous study by Lindberg et al. (2007), multiple
regression analyses was conducted to assess whether the distributions of urinary arsenic metabolites were dependent on sex, age, BMI,
selenium, and gene polymorphisms of GSTO1, AS3MT, and methylenetetrahydrofolate reductase (MTHFR). Here, we attempted to detect
the effects of sex, age, BMI, concentration of arsenic in water,
cumulative arsenic exposure status, and polymorphisms in GST
superfamily and AS3MT on arsenic level and profile in the Vietnamese
using a stepwise multiple regression analysis. Prior to the analysis, we
confirmed that there were no significant variations in sample
numbers of each sex, age and BMI in each genotype of GST
superfamily (p N 0.05). The multicollinearity of independent variables
for multiple regression analysis was examined by calculating the VIF.
The result showed that there was no significant multicollinearity (all
VIFs were less than 10).

The result of stepwise multiple regression analysis is shown in
Table 5. This multivariate assessment showed similar results to those
of univariate analysis in the present study (Tables 3 and 4) and our
previous results (Agusa et al., 2009b): associations between GSTO1
Glu155del and concentration of AsV in urine; between GSTP1
Ile105Val and concentrations of AB, MMAV, AsIII, IA and SA, %DMAV,
%AsIII, %IA, III/V and M/I in urine; between GSTM1 wild/null and %DMAV
in urine; between age and M/I; between sex and concentrations of
MMAV, AsIII and IA, %MMAV, %AsIII, %IA and D/M in urine, and
concentration of TA in hair; between BMI and concentrations of
DMAV, AsV, IA and SA in urine, and TA in hair; between concentrations
of TA in drinking water and hair; between AS3MT t4740c and
concentrations of DMAV, MMAV and IA, %AB, and %DMAV in urine;
between AS3MT t5913c and concentration of SA, %MMAV, and M/I
in urine; between AS3MT g12390c and %MMAV and D/M in urine;
between AS3MT t14458c and M/I in urine; between AS3MT g35991a

and %DMAV in urine; and between AS3MT g37853a and concentration
of AB, %AB, %DMAV, and %MMAV in urine. Furthermore, several
associations were newly identified by the multiple regression
analysis: associations of GSTO1 Glu155del with %AsV in urine; of
GSTM1 wild/null with D/M in urine; of age with DMAV level, SA level
and %DMAV in urine, and with TA level in hair; of sex with %AB and
M/I in urine; of AS3MT t4740c with AsIII level and M/I in urine; of
AS3MT t5913c with AsV level and D/M in urine; of AS3MT a6144t with
%MMAV in urine; of AS3MT g7395a with AsV level and %AsV in urine;
of AS3MT c14215t with III/V in urine; of AS3MT t35587c with AB and
MMAV levels in urine; and of AS3MT g35991a with M/I in urine.
Factors influencing metabolic capacity of arsenic were also characterized as follows: lower III/V in c/c homo type of AS3MT c14215t and
Ile/Val hetero type of GSTP1 Ile105Val; lower M/I in a/a homo type of

AS3MT g35991a, Ile/Ile homo type of GSTP1 Ile105Val, t/t homo type
of AS3MT t5913c, t/t homo type of AS3MT t14558c, male, younger
people, and t/t homo type of AS3MT t4740c; and lower D/M in g/g
homo type of AS3MT g12390c, male, null type of GSTM1, and c/c + t/
c types of AS3MT t5913c.
Although we statistically detected co-effects of genetic polymorphisms in GST superfamily as well as sex, age, BMI, TA in drinking water
and AS3MT genotypes on the arsenic concentration and metabolism,
adjusted determination coefficient (R2adj) in multiple regression equation was not so high (up to 0.344 for M/I) (Table 5). Thus, it seems
possible that there are other significant factors to account for the
variation in arsenic level and metabolism. Other SNP sites in GSTO1
(Mukherjee et al., 2006) and AS3MT (Wood et al., 2006), which are not
determined in the present study, might also be involved in the
variation. Mukherjee et al. (2006) found large variations of protein
expression levels of GSTO1 Cys32Tyr and GSTO2 Val41Ile, Cys130Tyr,
and L158Ile as well as those investigated in the present study (GSTO1
Ala140Asp, Glu155del, Glu105Lys, and Ala236Val, and GSTO2
Asn142Asp), indicating the possibility of significant variations in the
expression levels and catalytic functions among polymorphisms in
GSTO1 and GSTO2. Also, recombinant Arg173Trp and Thr306Ile
variants in AS3MT significantly suppressed levels of the enzyme
activity and immunoreactive protein compared to the wild type
(Wood et al., 2006). However, those variant allele frequencies are
quite low (up to 5%) in African-Americans, Caucasian-Americans, Han
Chinese-Americans, and Mexican-Americans (Mukherjee et al., 2006;
Wood et al., 2006), indicating the difficulty in confirming the
relationship among the population at small sample scale. Many SNPs
in the intron regions of GSTO1, GSTO2 and AS3MT have been reported
by Mukherjee et al. (2006), Wood et al. (2006), and NCBI SNP
Database, and the linkage between the intronic polymorphisms and
metabolism of arsenic is of interest. Alternatively, other genetic

polymorphisms such as MTHFR Ala222Val and Glu429Ala, which may
associate with arsenic metabolism in human (Lindberg et al., 2007;
Schläwicke Engström et al., 2007; Steinmaus et al., 2007), should be
considered. Further studies at larger scale are required to detect more
rigid relationships between genetic polymorphisms and arsenic
metabolism. Also, several nutritional factors such as β-carotene,
selenium and vitamins C and E are known to modify toxicity of arsenic
(Schoen et al., 2004) and thus such factors may influence arsenic
metabolism for residents in developing countries.
In summary, we suggest here that genotypes of GSTO1 Glu155del,
GSTP1 Ile105Val, and GSTM1 wild/null affect arsenic metabolism in a
Vietnamese population. Interestingly, GSTP1 Ile105Val polymorphism,
on which there is little information in association with arsenic
metabolism, showed statistically significant and wide associations
with urinary arsenic. In addition to the genetic polymorphisms of GST
superfamily, sex, age, BMI, TA in the drinking water, and various SNPs
in AS3MT were also related to arsenic level and profile in the Vietnamese. To our knowledge, this is the first comprehensive study
indicating the associations between genetic polymorphisms of GSTs
and arsenic metabolism in a Vietnamese population.


T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

Acknowledgments
We wish to thank Dr. A. Subramanian, CMES, Ehime University,
Japan for critical reading of the manuscript. The authors express
thanks to the staff of the CETASD, Hanoi University of Science and Dr.
H. Sakai, CMES (current affiliation; Laboratory of Structure-Function
Biochemistry, Department of Chemistry, Faculty of Science, Kyushu
University, Japan) for their help in sample collection. We also

acknowledge Ms. H. Touma and Ms. N. Tsunehiro, staff of the esBANK, CMES for their support in sample management and Ms. Y. Fujii,
Department of Legal Medicine, Shimane University Faculty of
Medicine, Japan (current affiliation; Department of Immunology,
Shimane University Faculty of Medicine, Japan) for her technical
assistant. This study was supported by Japan Society for the
Promotion of Science (JSPS) for the cooperative research program
under the Core University Program between JSPS and Vietnamese
Academy of Science and Technology (VAST). Financial support was
also provided by grants from Research Revolution 2002 (RR2002; to
H.I.) Project for Sustainable Coexistence of Human, Nature and the
Earth (FY2002), Grants-in-Aid for Scientific Research (S) (No.
20221003; to S.T.) and (A) (No. 19209025; to H.T.) from JSPS, and
21st Century and Global COE Programs from the Ministry of
Education, Culture, Sports, Science, and Technology (MEXT), Japan
and JSPS. The award of the JSPS Post Doctoral Fellowship for
Researchers in Japan to T. Agusa (No. 207871) is also acknowledged.
References
Agusa, T., Kunito, T., Kubota, R., Monirith, I., Tanabe, S., Tana, T.S., 2002. Arsenic
pollution in Cambodia. Biomed. Res. Trace Elem. 13, 254–255 (in Japanese).
Agusa, T., Kunito, T., Fujihara, J., Kubota, R., Minh, T.B., Trang, P.T.K., Subramanian, A.,
Iwata, H., Viet, P.H., Tanabe, S., 2004. Contamination by trace elements in
groundwater of Vietnam. Biomed. Res. Trace Elem. 15, 339–341.
Agusa, T., Inoue, S., Kunito, T., Kubota, R., Minh, T.B., Trang, P.T.K, Subramanian, A.,
Iwata, H., Viet, P.H., Tanabe, S., 2005. Widely-distributed arsenic pollution in
groundwater in the Red River Delta. Vietnam. Biomed. Res. Trace Elem. 16,
296–298.
Agusa, T., Kunito, T., Fujihara, J., Kubota, R., Minh, T.B., Trang, P.T.K., Iwata, H.,
Subramanian, A., Viet, P.H., Tanabe, S., 2006. Contamination by arsenic and other
trace elements in tube-well water and its risk assessment to humans in Hanoi.
Vietnam. Environ. Pollut. 139, 95–106.

Agusa, T., Kubota, R., Kunito, T., Minh, T.B., Trang, P.T.K., Chamnan, C., Iwata, H., Viet, P.H.,
Tana, T.S., Tanabe, S., 2007. Arsenic pollution in groundwater of Vietnam and
Cambodia: a review. Biomed. Res. Trace Elem. 18, 35–47.
Agusa, T., Fujihara, J., Takeshita, H., Iwata, H., Minh, T.B., Trang, P.T.K., Viet, P.H., Tanabe,
S., 2008. Relationship between genetic polymorphism of arsenic (+3 oxidation
state) methyltransferase (AS3MT) and profiles of urinary arsenic compounds in
Vietnamese. Biomed. Res. Trace Elem. 19, 265–267.
Agusa, T., Kunito, T., Minh, T.B., Kim Trang, P.T., Iwata, H., Viet, P.H., Tanabe, S., 2009a.
Relationship of urinary arsenic metabolites to intake estimates in residents of the
Red River Delta. Vietnam. Environ. Pollut. 157, 396–403.
Agusa, T., Iwata, H., Fujihara, J., Kunito, T., Takeshita, H., Minh, T.B., Trang, P.T.K., Viet, P.H.,
Tanabe, S., 2009b. Genetic polymorphisms in AS3MT and arsenic metabolism in
residents of the Red River Delta. Vietnam. Toxicol. Appl. Pharmacol. 236, 131–141.
Agusa, T., Inoue, S., Kunito, T., Minh, T.B., Ha, N.N., Tu, N.P.C., Trang, P.T.K., Iwata, H., Viet,
P.H., Tuyen, B.C., Tanabe, S., 2009c. Human exposure to arsenic from groundwater
in the Red River and the Mekong River Deltas in Vietnam. Int. J. Environ. Stud. 66,
49–57.
Aposhian, H.V., Aposhian, M.M., 2006. Arsenic toxicology: five questions. Chem. Res.
Toxicol. 19, 1–15.
Board, P.G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L.S., Schulte, G.K.,
Danley, D.E., Hoth, L.R., Griffor, M.C., Kamath, A.V., Rosner, M.H., Chrunyk, B.A.,
Perregaux, D.E., Gabel, C.A., Geoghegan, K.F., Pandit, J., 2000. Identification,
characterization, and crystal structure of the omega class glutathione transferases.
J. Biol. Chem. 275, 24798–24806.
Challenger, F., 1945. Biological methylation. Chem. Rev. 36, 315–361.
Chiou, H.Y., Hsueh, Y.M., Hsieh, L.L., Hsu, L.I., Yi-Hsiang, H., Hsieh, F.I., Wei, M.L., Chen,
H.C., Yang, H.T., Leu, L.C., Chu, T.H., Chen-Wu, C., Yang, M.H., Chen, C.J., 1997.
Arsenic methylation capacity, body retention, and null genotypes of glutathione
S-transferase M1 and T1 among current arsenic-exposed residents in Taiwan.
Mutat. Res. 386, 197–207.

Cullen, W.R., Reimer, K.J., 1989. Arsenic speciation in the environment. Chem. Rev. 89,
713–764.
Fujihara, J., Kunito, T., Takeshita, H., 2007. Frequency of two human glutathione-Stransferase omega-1 polymorphisms (E155 deletion and E208K) in Ovambo and
Japanese populations using the PCR-based genotyping method. Clin. Chem. Lab.
Med. 45, 621–624.

361

HapMap />Hayakawa, T., Kobayashi, Y., Cui, X., Hirano, S., 2005. A new metabolic pathway of
arsenite: arsenic-glutathione complexes are substrates for human arsenic
methyltransferase Cyt19. Arch. Toxicol. 79, 183–191.
Iwata, H., Kim, E.Y., Yamauchi, M., Inoue, S., Agusa, T., Tanabe, S., 2007. Chemical
contamination in aquatic ecosystems. Yakugaku Zasshi 127, 417–428 (in Japanese).
Kile, M.L., Houseman, E.A., Rodrigues, E., Smith, T.J., Quamruzzaman, Q., Rahman, M.,
Mahiuddin, G., Su, L., Christiani, D.C., 2005. Toenail arsenic concentrations, GSTT1
gene polymorphisms, and arsenic exposure from drinking water. Cancer Epidemiol.
Biomarkers. Prev. 14, 2419–2426.
Kubota, R., Kunito, T., Agusa, T., Fujihara, J., Monirith, I., Iwata, H., Subramanian, A., Tana,
T.S., Tanabe, S., 2006. Urinary 8-hydroxy-2′-deoxyguanosine in inhabitants
chronically exposed to arsenic in groundwater in Cambodia. J. Environ. Monit. 8,
293–299.
Lin, G.F., Du, H., Chen, J.G., Lu, H.C., Kai, J.X., Zhou, Y.S., Guo, W.C., Zhang, X.J., Lu, D.R., Golka,
K., Shen, J.H., 2007. Glutathione S-transferases M1 and T1 polymorphisms and arsenic
content in hair and urine in two ethnic clans exposed to indoor combustion of high
arsenic coal in Southwest Guizhou. China. Arch. Toxicol. 81, 545–551.
Lin, S., Shi, Q., Brent Nix, F., Styblo, M., Beck, M.A., Herbin-Davis, K.M., Hall, L.L.,
Simeonsson, J.B., Thomas, D.J., 2002. A novel S-adenosyl-L-methionine:arsenic(III)
methyltransferase from rat liver cytosol. J. Biol. Chem. 277, 10795–10803.
Lindberg, A.-L., Kumar, R., Goessler, W., Thirumaran, R., Gurzau, E., Koppova, K., Rudnai,
P., Leonardi, G., Fletcher, T., Vahter, M., 2007. Metabolism of low-dose inorganic

arsenic in a central European population: influence of sex and genetic polymorphisms. Environ. Health Perspect. 115, 1081–1086.
Loffredo, C.A., Aposhian, H.V., Cebrian, M.E., Yamauchi, H., Silbergeld, E.K., 2003.
Variability in human metabolism of arsenic. Environ. Res. 92, 85–91.
Mandal, B.K., Suzuki, K.T., 2002. Arsenic round the world: a review. Talanta 58,
201–235.
Marcos, R., Martinez, V., Hernandez, A., Creus, A., Sekaran, C., Tokunaga, H., Quinteros,
D., 2006. Metabolic profile in workers occupationally exposed to arsenic: role of
GST polymorphisms. J. Occup. Environ. Med. 48, 334–341.
Marnell, L.L., Garcia-Vargas, G.G., Chowdhury, U.K., Zakharyan, R.A., Walsh, B., Avram,
M.D., Kopplin, M.J., Cebrian, M.E., Silbergeld, E.K., Aposhian, H.V., 2003. Polymorphisms in the human monomethylarsonic acid (MMAV) reductase/hGSTO1
gene and changes in urinary arsenic profiles. Chem. Res. Toxicol. 16, 1507–1513.
McCarty, K.M., Chen, Y.C., Quamruzzaman, Q., Rahman, M., Mahiuddin, G., Hsueh,
Y.M., Su, L., Smith, T., Ryan, L., Christiani, D.C., 2007. Arsenic methylation, GSTT1,
GSTM1, GSTP1 polymorphisms, and skin lesions. Environ. Health Perspect. 115,
341–345.
Meza, M.M., Yu, L., Rodriguez, Y.Y., Guild, M., Thompson, D., Gandolfi, A.J., Klimecki, W.T.,
2005. Developmentally restricted genetic determinants of human arsenic metabolism: association between urinary methylated arsenic and CYT19 polymorphisms
in children. Environ. Health Perspect. 113, 775–781.
Mo, Z., Gao, Y., Cao, Y., Gao, F., Jian, L., 2009. An updating meta-analysis of the GSTM1,
GSTT1, and GSTP1 polymorphisms and prostate cancer: a HuGE review. Prostate
69, 662–688.
Mukherjee, B., Salavaggione, O.E., Pelleymounter, L.L., Moon, I., Eckloff, B.W., Schaid, D.J.,
Wieben, E.D., Weinshilboum, R.M., 2006. Glutathione S-transferase omega 1 and
omega 2 pharmacogenomics. Drug Metab. Dispos. 34, 1237–1246.
Naranmandura, H., Suzuki, N., Suzuki, K.T., 2006. Trivalent arsenicals are bound to
proteins during reductive methylation. Chem. Res. Toxicol. 19, 1010–1018.
Nordstrom, D.K., 2002. Worldwide occurrences of arsenic in ground water. Science 296,
2143–2145.
Paiva, L., Marcos, R., Creus, A., Coggan, M., Oakley, A.J., Board, P.G., 2008. Polymorphism
of glutathione transferase Omega 1 in a population exposed to a high

environmental arsenic burden. Pharmacogenet. Genomics 18, 1–10.
Schl ä wicke Engström, K.S., Broberg, K., Concha, G., Nermell, B., Warholm, M., Vahter,
M., 2007. Genetic polymorphisms influencing arsenic metabolism: evidence from
Argentina. Environ. Health Perspect. 115, 599–605.
Schmuck, E.M., Board, P.G., Whitbread, A.K., Tetlow, N., Cavanaugh, J.A., Blackburn, A.C.,
Masoumi, A., 2005. Characterization of the monomethylarsonate reductase and
dehydroascorbate reductase activities of Omega class glutathione transferase
variants: implications for arsenic metabolism and the age-at-onset of Alzheimer's
and Parkinson's diseases. Pharmacogenet. Genomics 15, 493–501.
Schoen, A., Beck, B., Sharma, R., Dubé, E., 2004. Arsenic toxicity at low doses: epidemiological
and mode of action considerations. Toxicol. Appl. Pharmacol. 198, 253–267.
Smedley, P.L., Kinniburgh, D.G., 2002. A review of the source, behaviour and distribution
of arsenic in natural waters. Appl. Geochem. 17, 517–568.
Sreeja, L., Syamala, V., Hariharan, S., Madhavan, J., Devan, S.C., Ankathil, R., 2005.
Possible risk modification by CYP1A1, GSTM1 and GSTT1 gene polymorphisms in
lung cancer susceptibility in a South Indian population. J. Hum. Genet. 50, 618–627.
Steinmaus, C., Moore, L.E., Shipp, M., Kalman, D., Rey, O.A., Biggs, M.L., Hopenhayn, C.,
Bates, M.N., Zheng, S.C., Wiencke, J.K., Smith, A.H., 2007. Genetic polymorphisms in
MTHFR 677 and 1298, GSTM1 and T1, and metabolism of arsenic. J. Toxicol.
Environ. Health A 70, 159–170.
Takeshita, H., Fujihara, J., Takastuka, H., Agusa, T., Yasuda, T., Kunito, T., 2009. Diversity
of glutathione S-transferase omega 1 (A140D) and 2 (N142D) gene polymorphisms
in worldwide populations. Clin. Exp. Pharmacol. Physiol. 36, 283–286.
Tanabe, S., 2006. Environmental Specimen Bank in Ehime University (es-BANK), Japan
for global monitoring. J. Environ. Monit. 8, 782–790.
Tanaka-Kagawa, T., Jinno, H., Hasegawa, T., Makino, Y., Seko, Y., Hanioka, N., Ando, M.,
2003. Functional characterization of two variant human GSTO 1-1s (Ala140Asp and
Thr217Asn). Biochem. Biophys. Res. Commun. 301, 516–520.
Vahter, M., 2000. Genetic polymorphism in the biotransformation of inorganic arsenic
and its role in toxicity. Toxicol. Lett. 112–113 209–217.

362

T. Agusa et al. / Toxicology and Applied Pharmacology 242 (2010) 352–362

Vahter, M., 2002. Mechanisms of arsenic biotransformation. Toxicology 181–182
211–217.
WHO, 2004. Guidelines for Drinking Water Quality, 3rd ed. World Health Organization,
Geneva, Switzerland.
Whitbread, A.K., Tetlow, N., Eyre, H.J., Sutherland, G.R., Board, P.G., 2003. Characterization of the human Omega class glutathione transferase genes and associated
polymorphisms. Pharmacogenetics 13, 131–144.
Wood, T.C., Salavagionne, O.E., Mukherjee, B., Wang, L., Klumpp, A.F., Thomae, B.A.,
Eckloff, B.W., Schaid, D.J., Wieben, E.D., Weinshilboum, R.M., 2006. Human arsenic
methyltransferase (AS3MT) pharmacogenetics: gene resequencing and functional
genomics studies. J. Biol. Chem. 281, 7364–7373.
Zakharyan, R.A., Aposhian, H.V., 1999. Enzymatic reduction of arsenic compounds in

mammalian systems: the rate-limiting enzyme of rabbit liver arsenic biotransformation is MMA(V) reductase. Chem. Res. Toxicol. 12, 1278–1283.
Zakharyan, R.A., Sampayo-Reyes, A., Healy, S.M., Tsaprailis, G., Board, P.G., Liebler, D.C.,
Aposhian, H.V., 2001. Human monomethylarsonic acid (MMA(V)) reductase is a
member of the glutathione-S-transferase superfamily. Chem. Res.Toxicol. 14,
1051–1057.
Zakharyan, R.A., Tsaprailis, G., Chowdhury, U.K., Hernandez, A., Aposhian, H.V., 2005.
Interactions of sodium selenite, glutathione, arsenic species, and omega class
human glutathione transferase. Chem. Res. Toxicol. 18, 1287–1295.
Zhong, S.L., Zhou, S.F., Chen, X., Chan, S.Y., Chan, E., Ng, K.Y., Duan, W., Huang, M., 2006.
Relationship between genotype and enzyme activity of glutathione S-transferases
M1 and P1 in Chinese. Eur. J. Pharm. Sci. 28, 77–85.