Toxicology and Applied Pharmacology 242 (2010) 352–362

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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

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

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

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))


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)



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)


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

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


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′

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



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:

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)


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


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)

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)

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)


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


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

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)


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

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

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


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


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


−0.299
−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

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


−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



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

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

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

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

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

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