DSpace at VNU: Individual variations in arsenic metabolism in Vietnamese: the association with arsenic exposure and GSTP1 genetic polymorphism

Cite this: Metallomics , 2012, 4, 91–100Individual variations in arsenic metabolism in Vietnamese: the association with arsenic exposure and GSTP1 genetic polymorphism Received 16th Augu

Cite this: Metallomics , 2012, 4, 91–100

Individual variations in arsenic metabolism in Vietnamese: the association with arsenic exposure and GSTP1 genetic polymorphism

Received 16th August 2011, Accepted 29th September 2011

DOI: 10.1039/c1mt00133g

We investigated the association of As exposure and genetic polymorphism in glutathione

S-transferase p1 (GSTP1) with As metabolism in 190 local residents from the As contaminated

groundwater areas in the Red River Delta, Vietnam Total As concentrations in groundwater

ranged fromo0.1 to 502 mg l1 Concentrations of dimethylarsinic acid (DMAV),

monomethylarsonic acid (MMAV), and arsenite (AsIII) in human urine were positively correlated

with total As levels in the groundwater, suggesting that people in these areas may be exposed to

As through the groundwater The concentration ratios of urinary AsIII/arsenate (AsV) and

MMAV/inorganic As (IA; AsIII+ AsV)(M/I), which are indicators of As metabolism, increased

with the urinary As level Concentration and proportion of AsIIIwere high in the wild type of

GSTP1Ile105Val compared with the hetero type, and these trends were more pronounced in the

higher As exposure group (456 mg l1creatinine in urine), but not in the lower exposure group

In the high As exposure group, AsIII/AsVratios in the urine of wild type of GSTP1 Ile105Val

were significantly higher than those of the hetero type, while the opposite trend was observed for

M/I These results suggest that the excretion and metabolism of IA may depend on both the As

exposure level and the GSTP1 Ile105Val genotype

It is well known that inorganic As (IA) is one of the human

carcinogenic chemicals Contamination by naturally derived IA

in groundwater has been reported in certain areas and has caused

a large number of serious health issues.1–3In such contaminated

areas, skin pigmentation, hyperkeratosis, cancers, and resultant

high mortalities have been caused by the chronic IA exposure in

the local people.4–6On the other hand, large differences in the

sensitivity to IA-related diseases among individuals have been

reported, suggesting its association with individual variations in

IA metabolism.7

Ingested IA is metabolized to methylated arsenicals in the body and then mainly excreted through urine There are two hypotheses regarding IA metabolic pathways;8 oxidative methylation9,10and reductive methylation.11,12In these metabolic pathways, two enzymes, arsenic (+3 oxidation state) methyl-transferase (AS3MT) and glutathione S-methyl-transferase o (GSTO), participate in the methylation and reduction of As compounds, respectively, in a variety of animals including the human.13 GSTs are a family of enzymes that play an important role in detoxification of various xenobiotics by catalyzing the conjugation of hydrophobic and electrophilic compounds with reduced glutathione There are seven classes of GSTs including

a, m, o, p, y, s, and z GSTO1 is involved in the reduction activities of arsenate (AsV), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV).14–16 DMAV reductase activity of GSTO2 is much lower than that of GSTO1.17 Some researchers have reported the relevance of genetic polymorphisms of GSTO1 and O2 to As metabolism

by in vitro assays17–19and in human studies.20–22

It has been suggested that GST p1 (GSTP1) plays a role in the reduction of IA toxicity An in vitro study using SA7 cells (As-resistant Chinese hamster ovary cells) revealed GSTP1 level-dependent resistance of IA.23 Zhou et al (2005) found that GSTP1 prevented IA-induced apoptosis in human lymphoma cell lines by reducing intracellular H2O2 levels.24 There are

a

Center for Marine Environmental Studies (CMES),

Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.

E-mail: iwatah@agr.ehime-u.ac.jp; Fax: +81-89-927-8172;

Tel: +81-89-927-8172

b

Department of Environmental Sciences, Faculty of Science,

Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan

c

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

d Department of Legal Medicine, Shimane University Faculty of

Medicine, Enya 89-1, Izumo 693-8501, Japan

e Vietnam Environment Administration (VEA),

Ministry of Natural Resources and Environment (MONRE),

273 Doi Can street, Ba Dinh, Hanoi, Vietnam

several single nucleotide polymorphisms (SNPs) in GSTP1

(http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId = 2950)

For the GST activity, the Ile (AA) type of GSTP1 Ile105Val

(rs1695; A to G substitution at nucleotide base 6624 and

isoleucine to valine substitution at amino acid base 105) was

higher than the Val (GG) type in the erythrocyte.25

Several epidemiological studies have recently investigated

the relationships between genetic polymorphisms in GSTP1

and IA-related diseases (Table 1), but the results were not

consistent In the study in As-contaminated areas of West

Bengal, no association of GSTP1 Ile105Val with skin lesion

was observed.26McCarty et al (2007) have reported that there

was no significant difference in the genotype distribution of

GSTP1 Ile105Val between Bangladeshi people with and

without skin lesion.27On the other hand, several studies have

suggested that the Val type of GSTP1 Ile105Val was

associated with increased risks of As-induced skin lesion in

Chinese28and Bangladeshi,29and of atherosclerosis in Taiwanese.30

Wang et al (2007) also reported that the risk increased in Val type

GSTP1 with high As exposure.30 However, Hsu et al (2008)

evaluated the interaction of GSTP1 polymorphism with urinary

transitional cell carcinoma in southwestern Taiwan and found that

the Val type of GSTP1 Ile105Val showed a significantly lower

cancer risk than the Ile type, suggesting that the wild type of

GSTP1 Ile105Val may be sensitive to urinary transitional cell

carcinoma.31

There are few studies evaluating whether GSTP1 Ile105Val

polymorphism influences As metabolism Marcos et al (2006)

revealed that the Val type had higher %DMAVthan the Ile

type, but it was not significant in Chilean.32 Since 2001, we

have investigated As pollution in groundwater and its human

exposure in Vietnam.8,33–43 Recently, we have focused on

the association of genetic polymorphisms in As metabolic

enzymes including GSTP1 with As metabolism and suggested

that the heterozygote of GSTP1 Ile105Val had a higher

metabolic capacity from IA to monomethyl As, while the

opposite trend was observed for the metabolism from AsVto

AsIII.40,41However, the metabolic capacity of As has not been

evaluated in association with GSTP1 Ile105Val polymorphisms

and As exposure levels Inconsistent findings among previous

studies on association of GSTP1 Ile105Val with As metabolic

capacity as well as As-related diseases may partly result from

the joint effects of human As exposure levels and genetic

polymorphisms in GSTP1

The aim of this study is to clarify the association of As

exposure and genetic polymorphism in GSTP1 Ile105Val with

As metabolism as well as their co-interaction Therefore, we

analyzed concentrations of As compounds in the urine and genotyped GSTP1 Ile105Val in residents from As-contaminated groundwater areas in Vietnam Biological factors such as sex, age, body mass index (BMI), and habits of alcohol consumption and smoking were also incorporated to evaluate their contributions to individual variations in IA metabolism

2.1 Samples Samples of well water (n= 64) and human urine (n= 190) and blood (n= 190) were randomly collected from Hoa Hau (HH) and Liem Thuan (LT) in Ha Nam Province in March 200639 and from Thanh Vanh (TV) and THach Hoa (TH) in Ha Tay Province in September 2007 All communes are located in the rural area of the Red River Delta, Vietnam and there are

no other significant sources of As such as mining sites or industries Several houses in HH, LT, and TV had wells equipped with a sand filter system and thus the sand-filtrated groundwater samples (n= 40) were also collected along with unfiltered water samples Usage period of the wells and their depth are shown in Table 2 The informed consent was obtained from all the participants This study has been approved by the Ethical Committee of Ehime University, Japan Information on sex, age, residential years, body height and weight, body mass index (BMI), and smoking and alcohol habits of donors is summarized in Table 2 All samples were kept at 25 1C in a freezer of the Environmental Specimen Bank (es-BANK) in Ehime University44until chemical analyses and genotyping

2.2 Analyses of As Analytical methods for samples collected in 2006 have already been reported in our previous study.39 The methods of As analysis of water, and human urine and blood samples collected in 2007 were briefly summarized below After acidification with concentrated HNO3, total As (TA) in water samples was analyzed with an inductively coupled plasma-mass spectro-meter (ICP-MS; HP-4500, Hewlett-Packard, Avondale, PA, USA) Rhodium was used as an internal standard to correct matrix effects and instrumental drift.39Accuracy of the analytical method was confirmed in good agreement (91–95%) with certified

TA concentration by analyzing a certified reference material, SLRS-4 River Water from the National Research Council Canada (NRCC) In addition, we have participated in an inter-calibration exercise program organized by the Swiss Federal Institute of

Table 1 Associations of GSTP1 Ile105Val polymorphism with endpoints related to As

Taiwan Atherosclerosis High risk in Val type under high As exposure 30

Aquatic Science and Technology (Eawag) in the frame of

the ongoing cooperation of Vietnam and Switzerland for

As-related researches Concentration of TA in water samples

is expressed in mg per l

Urinary As compounds including arsenobetaine (AB),

DMAV, MMAV, AsIII, and AsV were separated by a

high-performance liquid chromatograph (HPLC; Shimadzu,

LC10A Series, Kyoto, Japan) equipped with an Inertsil AS

column (15 cm, 2.1 mm i.d.; GL Sciences Inc., Japan) The

column was equilibrated with the mobile phase (10 mM

sodium 1-butanesulfonate, 4 mM tetramethylammonium

hydroxide, 4 mM malonic acid, and 0.5% methanol; pH 3.0

was adjusted with nitric acid) at a flow rate of 0.5 ml min1at

45 1C As internal standard, Rh was added into the buffer The

injection volume was 10 ml Five arsenicals separated by the

column were determined with ICP-MS Ion intensities at m/z

75 (75As), 77 (40Ar37Cl and 77Se), and 103 (103Rh) were

monitored and there was no interference during HPLC/ICP-MS

analysis A certified reference sample, NIES No 18 human urine

that was provided by the National Institute for Environmental

Studies (NIES), Japan, was analyzed to assure the methodological

accuracy Analyzed concentrations of AB and DMAVwere in

good agreement with the certified values (90–106%) In the

present study, sum of all As compounds, inorganic As

(AsIII+ AsV), and AsIII+ AsV+ MMAV+ DMAVdetected

in urine sample are denoted as SA, IA, and IMDA, respectively

Percentages of AB, AsIII, AsV, MMAV, DMAV, IA, and IMDA

to SA in the human urine were denoted as %AB, %AsIII,

%AsV, %MMAV, %DMAV, %IA, and %IMDA, respectively

Urinary creatinine was determined at SRL, Inc (Tokyo, Japan)

and concentrations of As compounds in the urine were expressed

as mg As per g on a creatinine basis Because AsV, IA, and MMAV are metabolized to AsIII, MMAV, and DMAV, respectively, in the human body, concentration ratios of AsIII/AsV(III/V), MMAV/

IA (M/I), and DMAV/MMAV(D/M) were used as an index for each metabolic process of AsV, IA, and MMAV

2.3 Genotyping of GSTP1 polymorphisms Genotyping of GSTP1 followed the methods described in our previous study.40A QIAamp DNA mini kit (Qiagen, Chatworth, CA) was used to extract DNA from whole blood sample The reference sequence of GSTP1 (accession number, AY324387) was based on the DNA Data Bank of Japan (DDBJ) Forward and reverse primers of GSTP1 Ile105Val were 50

-TGAGGGCACAA-GAAGCCCCT-30, respectively DNA was amplified with PCR

in a 10 ml reaction mixture containing GoTaqs

Green Master Mix (Promega, Madison WI, USA) at 55 1C of annealing temperature and then treated with Bsm AI at 37 1C The PCR products were separated in 8% polyacrylamide gel by electro-phoresis (300 V, 15 min) and were detected by silver staining The genotyping was carried out in duplicate The representativeness

of nucleotide sequences for the genotype was confirmed with a Genetic Analyzer (model 310, Applied Biosystems)

2.4 Statistical analyses Together with the data in the present study, analytical data in the water and human urine from HH and TL provided in our previous study39,40were used for statistical analyses StatView

Table 2 Information on water and human samples from Hoa Hau (HH), Liem Thuan (LT), Thanh Vanh (TV), and Thach Hoa (TH) in Vietnam

Groundwater

Used period (years) a 9 (5.5–13) 6 (1–16) 7 (3–12) 6 (3–10) 0.015 c Well depth (m)a 14 (8–16) 15 (12–24) 38 (20–60) 33 (24–50) o0.001 c

TA (mg l 1 ) b 368 (163–502, and 2120 (an outlier)) 1.4 (0.7–6.8) 36.0 (5.5–145) 0.1 ( o0.1–0.5) o0.001 c Filtered water

TA (mg l1)b 18.9 (3.2–143) 2.0 (1.0–4.9) 5.4 (1.5–50.7) — o0.001 c Drinking water e

TA (mg l1) b 50.1 (3.2–486) 1.7 (0.9–4.9) 5.4 (1.5–50.7) 0.1 ( o0.1–0.5) o0.001 c Subjects

Age (years)a 37 (11–60) 34 (11–70) 32 (13–71) 35 (15–60) 40.05c Residential time (years) a 33 (3–60) 31 (6–65) 30 (13–71) 17 (3–45) o0.001 c Height (cm)a 156 (137–173) 150 (121–169) 155 (142–170) 158 (137–171) o0.001 c Weight (kg) a 48 (27–66) 44 (22–67) 46 (32–65) 52 (38–72) 0.001 c

No of alcohol drinker/non-alcohol drinker 14/37 10/39 13/37 13/27 40.05d Urinary SA (mg g:1creatinine) b 92.6 (45.2–365) 97.9 (38.6–397) 63.5 (28.7–115) 43.2 (20.0–96.0) o0.001 c Urinary AB (%) a 22.7 (4.0–56.8) 19.6 (3.1–58.6) 16.0 (0–63.5) 28.3 (3.0–78.1) 0.001 c Urinary DMAV(%)a 55.9 (32.6–77.2) 59.0 (29.1–78.9) 51.8 (26.1–68.6) 44.4 (13.1–68.8) o0.001 c Urinary MMA V (%) a 10.6 (2.9–17.8) 10.0 (4.8–20.9) 11.5 (3.3–20.1) 7.2 (0–15.6) o0.001 c Urinary AsIII(%)a 8.5 (0–20.3) 8.7 (0–19.8) 9.7 (0–30.0) 6.6 (0–16.6) 0.013c Urinary As V (%) a 2.3 (0–11.1) 2.7 (0–11.3) 11.0 (3.1–34.4) 13.5 (0–37.4) o0.001 c

a Arithmetic mean and range b Geometric mean and range c Tukey–Kramer test d w 2 test e In a house equipped with sand filter, filtered water instead of raw groundwater is assumed to be consumed.

(version 5.0, SASs

Institute, Cary, NC, USA), PASW Statistics (v 18.0J, SPSS Inc., Chicago, IL, USA), and EXCEL Toukei

(Version 6.05, Esumi Co., Ltd., Tokyo, Japan) were used for the

statistical analyses One half of the value of the respective limits

of detection was substituted for those values below the limits

of detection and used in the statistical analysis Normality of

the distribution of all variables was checked by Kolmogorov–

Smirnov’s one sample test To adapt parametric analyses, the

data, which showed non-normal distribution, were

log-trans-formed Student’s t-test and the Tukey–Kramer test were

conducted to find differences in As levels and compositions

in human urine among locations and the genotype of GSTP1

A w2test was employed for checking sample size distribution in

each group category Relationships between variables were

assessed by the Pearson correlation coefficient To assess the

factors affecting As levels in the urine and metabolic capacity

of As, a stepwise multiple regression analysis was executed In

the regression models, nominal variables such as As exposure

status, sex, alcohol and smoking habits, and genotype of

GSTP1 Ile105Val were transformed to dummy variables

(0 and 1) The multicollinearity of independent variables was

assessed by calculating the variance inflation factor (VIF)

po 0.05 was considered to be statistically significant

Concentration of TA in groundwater

Concentrations of TA in groundwater are shown in Table 2

The range of concentration waso0.1–502 mg l1(one sample

that had 2120 mg l1was regarded as an outlier and removed

from further statistical analysis because the water had large

amounts of particles) A significant regional difference in TA

concentration was observed; HH (geometric mean (GM),

368 mg l1) 4 TV (GM, 36.0 mg l1) 4 LT (GM, 1.4 mg l1) 4

TH (GM, 0.1 mg l1) (po 0.001) 54.7% of all groundwater

samples exceeded the drinking water guideline (10 mg l1)

established by WHO.5 Remarkably, 100% and 95.2% of

groundwater samples from HH and TV had TA concentration

over the guideline value.5These results indicate that

ground-waters from HH and TV are not suitable for drinking

Analyses of relationships between TA concentration and well

depth showed a significant positive correlation in the

ground-water from TV (r= 0.693, p= 0.003), indicating that the

concentration of TA may be higher in the deeper layer of the

aquifer in TV

Several residents have consumed sand-filtered groundwater

in these sampling areas except in TH Concentrations of TA in

the filtered water were in the range of 1.0–143 mg l1(Table 2)

There was a significant (po 0.001) regional difference in TA

concentrations in the filtered water; HH (GM, 18.9 mg l1) 4

TV (GM, 5.4 mg l1) 4 LT (GM, 2.0 mg l1) Through the

sand filtration, TA concentrations in the raw groundwater

from HH and TV significantly reduced (po 0.001) and the

removal efficiencies were 93% in HH and 82% in TV on

arithmetic mean (AM) (Fig 1) However, 80% and 29% of

filtered-water samples from HH and TV were still higher than

the WHO guideline value.5 This result suggests that safe

drinking water is not always obtained by only a sand filter

system and thus, further removal techniques of As from groundwater are required in highly As-contaminated groundwater areas

To evaluate the As exposure status of the residents, we considered the well water, which local people are drinking, as the major source of As Concentration of TA in drinking water was regarded as those in raw groundwater for the houses without a sand-filter system, and as those in filtered water for the houses with the filter system Concentrations of TA in drinking water from HH, TV, LT, and TH are shown in Table 2 The highest As concentration in drinking water was observed in HH (GM, 50.1 mg l1), followed by TV (GM, 5.4 mg l1), LT (GM, 1.7 mg l1), and TH (GM, 0.1 mg l1) and a significant difference was detected among all the four locations (po 0.001) In HH and TV, samples with TA concen-trations exceeding the guideline value for drinking water5were 88% and 29%, respectively Considered that As concentration in the drinking water represents close to the real exposure status

in local residents, potential health risk of people drinking those As-contaminated water is of great concern

Concentration and composition of As compounds in human urine Concentrations of SA and composition of As compounds in the urine of people from HH, TV, TL, and TH are summarized in Table 2 Urinary As was detected in all samples and the range of urinary SA concentrations was from 20.0 to 397 mg g1 creatinine

To understand the exposure level of As in local people through drinking water, relationships between As concentra-tions in drinking water and human urine were assessed As shown in Fig 2, concentrations of DMAV (R2 = 0.118,

po 0.001), MMAV

(R2 = 0.141, po 0.001), and AsIII (R2= 0.068, po 0.001) in human urine were positively correlated with that of TA in drinking water Significant positive correlations between concentrations of TA in drinking water and urinary AsV (R2= 0.028, p = 0.036), IMDA (R2= 0.114, po 0.001), and SA (R2 = 0.088, po 0.001) were also observed (data not shown) These results suggest that the residents in these areas are exposed

to As through the consumption of drinking water and ingested

Fig 1 Concentrations of TAs in raw and sand-filtrated groundwater from Hoa Hau (HH), Thanh Vanh (TV), and Liem Thuan (LT)

in Vietnam Bar indicates each concentration of TA in raw and sand-filtered groundwater.

As are metabolized to MMAVand DMAV in the body On

the other hand, concentration of urinary AB, which is

probably derived from fish and shellfish ingestion, showed

no association with the TA level in drinking water (p4 0.05)

(Fig 2)

Among the As compounds detected, DMAV (AM, 53%)

was the most predominant species, followed by AB (AM,

21%), MMAV (AM, 10%), AsIII(AM, 8%), and AsV(AM,

7%) Because As compounds are transformed by reduction

and methylation processes in the human body,9–12

concen-tration ratios of AsIII/AsV (III/V), MMAV/IA (M/I), and

DMAV/MMAV (D/M) in human urine were defined as

metabolic indices for the reduction, first methylation, and

second methylation, respectively In the present study, GM

for III/V, M/I, and D/M were 1.2, 0.7, and 5.4, respectively

Genotype distribution of GSTP1 Ile105Val

Genotyping results of GSTP1 Ile105Val showed no mutation

of the homo type (Val/Val) in this population Genotype

frequency in all donors was 0.68 for AA (Ile/Ile) and

0.32 for AG (Ile/Val), whereas A and G allele frequencies were

0.84 and 0.16, respectively However, the GSTP1 Ile105Val

genotype did not follow the Hardy–Weinberg principle in this

study (p= 0.010) Although the reason remains unclear, we

could confirm no genotyping error by conducting duplicate

analyses of all DNA samples and by sequence analyses of some

representative samples Compared with the allele frequency of

GSTP1Ile105Val in 11 populations published in the HapMap

database (HapMap Data Rel 28 PhaseII +III, August 10, on

NBCI B36 assembly, dvSNP b 126; http://hapmap.ncbi.nlm

nih.gov/cgi-perl/snp_details_phase3?name = rs1695&source =

hapmap28_B36&tmpl = snp_details_phase3), the A allele

frequency (0.84) in Vietnamese detected in this study was

similar to those in Chinese populations (0.816 in Han Chinese

in Beijing, China groups (CHB (H)) and 0.812 in Chinese in

Metropolitan Denver, Colorado (CHD (D)) On the contrary,

allele distribution in Vietnamese in the present study was

largely different from Africans, Europeans, and Americans (0.448–0.701 for A allele frequencies)

Factors influencing As concentration and metabolism in humans

To understand which factors can affect the concentration of

As and its metabolic capacity, a stepwise multiple regression analysis was performed As potential factors, As exposure status, genotype of GSTP1 Ile105Val, sex, age, BMI, alcohol consumption, and smoking habit were taken into considera-tion Before the analyses, the As exposure level was defined by dividing all donors into two categories, high (HA) and low (LA) As exposure groups, based on GM for urinary IMD concentration (56 mg g1 creatinine) No significant bias in sample numbers among As exposure level, genotype of GSTP1 Ile105Val, and sex was validated by a w2test Sex ratios were significantly different in both smoking and alcohol habits, because only a few females had these habits The calculated VIF values of explanatory variables were less than 10, and thus multi-collinearity in the multiple regression analysis was rejected

Results of the multiple regression analyses are listed in Table 3 When all donors were evaluated, As exposure level, genotype of GSTP1 Ile105Val, sex, and BMI were significantly correlated with urinary As concentration and metabolic capacity, with the influence of exposure status being the strongest These results were similar to our previous study.40 Remarkably, the exposure level of As was significantly associated with not only urinary concentrations of As compounds

as expected, but also metabolic indices except for D/M Indicators

of As metabolism such as %DMAV, %MMAV, %AsIII,

%IMDA, III/V, and M/I of HA were higher than those of LA, while the opposite results were observed for %AB, %AsV, and

%IA (Table 3) Comparisons of III/V and M/I between HA and

LA are shown in Fig 3 These results indicate that the metabolism from AsVto AsIIIand from IA to MMAVmay be facilitated by high As exposure level No significant increase in D/M with the

As exposure level implies that 2nd methylation may not be facilitated by high exposure Although decreased %AB could

be explained by increased %DMAV, %MMAV, and %IA with

As exposure, it was not clear why higher concentration of urinary

AB was observed

In all participants, GSTP1 Ile105Val was associated with III/V, M/I, and concentrations of AB, AsIII, IA, IMDA, and

SA Negative correlations between BMI and concentrations of MMAV, AsV, IA, and IMDA in human urine were observed Females had higher %DMAVand D/M than males

Because it was found that the As exposure status signifi-cantly influenced many parameters (Table 3), we repeated the stepwise regression analysis by dividing all donors into

HA and LA to better understand the difference in factors associated with As excretion and metabolic capacity between those groups (Table 4)

Interestingly, it was found that the factors, which could relate to As concentration and metabolism, were different between HA and LA except relationships between sex and D/M (Table 4); D/M in females was significantly higher than that in males regardless of the As exposure level, suggesting a higher methylation capacity from MMAVto DMAVin females

Fig 2 Relationships between concentrations of TA in drinking water

and As compounds (DMA V , MMA V , As III , and AB) in human urine

from Hoa Hau (HH), Thanh Vanh (TV), Liem Thuan (LT), and

Thach Hoa (TH) in Vietnam Dashed line indicates WHO guideline

value (10 mg l1) for drinking water (WHO, 2004).

regardless of the As exposure level It has been reported that

the 2nd methylation capacity is higher in females than in males

in most studies.39,45Our results support these previous reports

The present study also showed that %MMAV in males was

significantly higher compared with females in HA A similar

trend was observed for %AsVin LA, although the significant

level was weak The sexual difference in methylation capacity

may be partly associated with an estrogen-related metabolic pathway.45 The mechanism of sexual difference in As metabolism needs more attention in future studies

The GSTP1 Ile105Val hetero type had lower concentrations

of AsIII, IA, and IMDA, and III/V and higher M/I than the wild type in HA, but not in LA (Table 4) For urinary concentration of AsIII(Fig 4), it is suggested that the GSTP1 genotype may be linked with the excretion of AsIIIinto the urine Leslie et al (2004) investigated a transport mechanism

of AsIIIby a multidrug resistance protein 1 (MRP1/ABCC1) using a specific cell line, H69AR over-expressing MRP1 and found that MRP1 can transport AsIII only in the presence of GSH and expression of GSTP1 in the plasma membrane is required for the transportation of AsIII(+GSH).46Zhong et al (2006) reported that in the erythrocyte of the healthy Chinese, the GSTP1 Ile105Val wild type showed a higher catalytic activity than the mutation type.25Considering these reports together, in the higher As exposure group, the GSTP1 Ile105Val wild type might accelerate the conjugation of GSH to AsIIImore than the mutation type and the conjugate may be more efficiently excreted from the cell through the MRP1 transporter Further in vivo and human case studies are needed to verify this hypothesis

Table 3 Stepwise multiple regression analysis of As concentrations and compositions in urine against sex a , age, BMI, alcohol and smoking habitsa, As exposure level, and polymorphism of GSTP1 Ile105Vala

Dependent

variable R2adj p Independent variable b p

Dependent variable R2adj p Independent variable b p

%AB 0.031 0.015 Exposure

(0 = low, 1 = high)

0.193 0.015 AB 0.085 o0.001 Exposure

(0 = low, 1 = high)

0.254 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.156 0.027

%DMAV 0.094 o0.001 Exposure

(0 = low, 1 = high)

0.287 o0.001 DMA V

0.564 o0.001 Exposure

(0 = low, 1 = high)

0.753 o0.001 Sex (0 = female, 1 = male) 0.158 0.039

%MMA V 0.028 0.021 Exposure

(0 = low, 1 = high)

0.183 0.021 MMA V 0.410 o0.001 Exposure

(0 = low, 1 = high)

0.613 o0.001

%AsIII 0.016 0.045 Exposure

(0 = low, 1 = high)

0.146 0.045 AsIII 0.258 o0.001 Exposure

(0 = low, 1 = high)

0.472 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.173 0.007

%As V 0.080 o0.001 Exposure

(0 = low, 1 = high)

0.293 o0.001 As V 0.039 0.009 BMI 0.188 0.010

Exposure (0 = low, 1 = high)

0.143 0.049

%IA 0.025 0.026 Exposure

(0 = low, 1 = high)

0.177 0.026 IA 0.250 o0.001 Exposure

(0 = low, 1 = high)

0.393 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.217 o0.001

%IMDA 0.031 0.015 Exposure

(0 = low, 1 = high)

0.193 0.015 IMDA 0.627 o0.001 Exposure

(0 = low, 1 = high)

0.757 o0.001

GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

0.104 0.021 III/V 0.196 o0.001 Exposure

(0 = low, 1 = high)

0.402 o0.001 SA 0.510 o0.001 Exposure

(0 = low, 1 = high)

0.693 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.183 0.019 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.139 0.007 M/I 0.144 o0.001 Exposure

(0 = low, 1 = high)

0.370 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.159 0.021 D/M 0.057 o0.001 Sex (0 = female, 1 = male) 0.250 o0.001

a These nominal variables were transformed to dummy variables (0 or 1).

Fig 3 Comparison of III/V and M/I between low (LA) and high As

(HA) exposure groups from Hoa Hau (HH), Thanh Vanh (TV), Liem

Thuan (LT), and Thach Hoa (TH) in Vietnam Data are given as

geometric mean and geometric standard deviation *** indicates

statistical significance at p o 0.001.

A lower III/V in the hetero type of GSTP1 Ile105Val than

the wild type in all participants and HA (Table 3 and 4, and

Fig 5) suggest that the heterozygote of GSTP1 Ile105Val

might have a lower reduction capacity from AsVto AsIIIand

this reduction capacity may depend on the As exposure level

Although the reductase activity of AsV by GSTP1 was not

measured, the wild GSTP1 Ile105Val protein has a high

catalytic GST activity compared with the mutation type.25

The hetero type of GSTP1 Ile105Val had higher M/I than

the wild type in all participants and HA (Fig 5) This result

may be associated with the capacity of As transport from cells,

because GSTP1 has no function of As methylation Considering that the GSTP1 Ile105Val hetero type may have less function to

Table 4 Stepwise multiple regression analysis of As concentrations and compositions in urine against sex a , age, BMI, alcohol and smoking habitsa, As exposure statusa, and polymorphism of GSTP1 Ile105Valafor each As exposure level

Dependent

variable R2adj p Independent variable b p

Dependent variable R2adj p Independent variable b p High As exposure

DMA V 0.085 0.005 BMI 0.397 0.001

%MMAV 0.057 0.009 Sex (0 = female, 1 = male) 0.258 0.009 MMAV 0.048 0.016 BMI 0.240 0.016

%As III 0.066 0.006 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.274 0.006 As III 0.126 o0.001 GSTP1

(0 = Ile/Ile, 1 = Ile/Val)

0.367 o0.001

IA 0.179 o0.001 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.377 o0.001

GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

0.242 0.010

III/V 0.068 0.019 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.287 0.019 SA 0.051 0.014 BMI 0.245 0.014 M/I 0.121 0.001 Age 0.264 0.007

GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val)

0.249 0.010 D/M 0.042 0.024 Sex (0 = female, 1 = male) 0.227 0.024

Low As exposure

AB 0.054 0.016 GSTP1 Ile105Val

(0 = Ile/Ile, 1 = Ile/Val)

0.255 0.016

%DMA V 0.066 0.008 Sex (0 = female, 1 = male) 0.277 0.008 DMA V 0.057 0.013 Sex (0 = female, 1 = male) 0.261 0.013

%AsV 0.035 0.044 Sex (0 = female, 1 = male) 0.214 0.044

%IA 0.038 0.038 BMI 0.220 0.038

D/M 0.133 0.001 Sex (0 = female, 1 = male) 0.298 0.004

a These nominal variables were transformed to dummy variables (0 or 1).

Fig 4 Comparison of As III concentration between the wild and

hetero types of GSTP1 Ile105Val among all participants, and high

(HA) and low As (LA) exposure groups from Hoa Hau (HH), Thanh

Vanh (TV), Liem Thuan (LT), and Thach Hoa (TH) in Vietnam Data

are given as geometric mean and geometric standard deviation ** and

*** indicate statistical significance at po 0.01 and po 0.001,

respectively.

Fig 5 Comparison of III/V and M/I between the wild and hetero types of GSTP1 Ile105Val among all participants, and high (HA) and low As (LA) exposure groups from Hoa Hau (HH), Thanh Vanh (TV), Liem Thuan (LT), and Thach Hoa (TH) in Vietnam Data are given as geometric mean and geometric standard deviation * and ** indicate statistical significance at po 0.05 and po 0.01, respectively.

excrete AsIII(IA) (Fig 4), the pathway of methylation from IA

to MMAVmight be more dominant than the excretion in the

hetero type In addition, this may be more likely when people

are exposed to high As In the copper mine workers from Chile,

%DMAVin the Val type of GSTP1 Ile105Val was higher than

that in the Ile type, although the result was not statistically

significant32 (Table 1) Similarly, this study revealed no

association of %DMAVin the GSTP1 Ile105Val genotype

BMI has been used as an indicator of nutritional status or

obesity By using the stepwise regression analyses, BMI had

negative correlations with DMAV, MMAV, IA, IMDA, and

SA concentrations in HA (Table 4) These results suggest two

hypotheses; the exacerbation of nutritional status by As

exposure and the effect of increased body fat on As accumulation

in the high As exposure group Similar results were obtained in

Vietnam in our previous studies.39,40Other studies47–50have

reported the interaction between BMI and metabolic capacity

of As, which was not observed in our studies In the present

study, a negative correlation between BMI and %IA was

found only in LA Increased %DMAV and decreased

%MMAV with an increase in BMI in local residents

were reported from blackfoot disease-hyperendemic areas in

Taiwan47and in European males.48On the other hand, there

are some contradictory reports, indicating no significant

association of BMI with As metabolism.49,50

Age was positively correlated with M/I and concentrations

of DMAVand IMDA only in HA (Table 4) Similar findings

were reported in Vietnamese.39,40Kurttio et al (1998) found a

slight increase of DMAVwith age in adults from Finland.51In

a study of Argentina, %IA decreased with age, but there were

no age-dependent variations in %MMAV, %DMAV, and

D/M.52It has been suggested that children may have a higher

2nd methylation capacity compared to adults.37,53,54However,

no clear associations were detected between age and urinary

D/M or %DMAVin the present study, probably due to small

sample size of children (n= 21 foro 15 years old)

One should notice that adjusted determination coefficients

(R2adj) in the multiple regression equations were moderate

(0.016–0.627), even though the p values were less than 0.001

This suggests that there are other factors that are involved in

As concentration and metabolism of the participants Genetic

polymorphisms of other As metabolic enzymes such as

AS3MT8,39,40,41 and methylenetetrahydrofolate reductase

(MTHFR)48,52,55 may be one of the potential factors In

addition, several SNPs are known in MRP1.56Further studies

are necessary to assess potential effects of these genetic variations

on the metabolism and toxicity of As

This study revealed that both environmental (As exposure

status) and genetic factors (GSTP1 Ile105Val polymorphism)

are significantly associated with the concentration and

metabolism of As in humans Furthermore, it can be suggested

that the association of GSTP1 Ile105Val polymorphism with

As is enhanced under high As exposure This means that it can

be important when association of genetic polymorphisms in

As metabolic enzymes is evaluated in some populations

A proposed mechanism of As metabolism and excretion by GSTP1 Ile105Val is summarized in Fig 6 The wild type (Ile type) of GSTP1 Ile105Val may have a high reductive capacity from AsV to AsIII GSTP1 may conjugate GSH to

AsIII on the membrane and then AsIII-GS may be excreted through MRP1 Since the GSTP1 Ile105Val wild type may have a higher activity than the hetero type, the wild type may efficiently excrete AsIII-GSH compared with the hetero type On the contrary, the hetero type (Val type) of GSTP1 Ile105Val may have a relatively lower reduction activity and excretion and thus the metabolism to MMAVmay be more facilitated These pathways may be accelerated with an increase in the As exposure status in humans To verify these hypotheses, further studies are required to determine (i) whether GSTP1 can reduce from AsIIIto AsVlike GSTO1; (ii) whether GSTP1 can facilitate excretion of AsIIIthrough MRP1; (iii) whether these functions vary among the GSTP1 Ile105Val genotype; and (iv) whether interaction of GSTP1 polymorphism with As excretion and metabolism is influenced by the As exposure level

Abbreviations

AS3MT As (+3 oxidation state) methyltransferase

AsIII arsenite

DMAV dimethylarsinic acid Eawag Swiss Federal Institute of Aquatic Science and

Technology GST glutathione-S-transferase GSTO1 glutathione-S-transferase o 1 GSTO2 glutathione-S-transferase o 2 GSTP1 glutathione-S-transferase p 1

HPLC high performance liquid chromatograph

IA inorganic As Here, As[V]+ As[III]

ICP-MS inductively coupled plasma mass spectrometer III/V AsIII/AsV

IMDA AsIII+ AsV+ MMAV+ DMAV

Fig 6 Suspected pathways of As metabolism and excretion by GSTP1 Ile105Val polymorphism Solid and dashed arrows indicate strong and weak pathways, respectively.

M/I MMAV/IA

MMAV monomethylarsonic acid

MRP1 multidrug resistance protein 1

MTHFR methylenetetrahydrofolate reductase

NRCC National Research Council Canada

PCR-RFLP PCR restriction fragment length polymorphism

SNP single nucleotide polymorphism

VIF variance inflation factor

Acknowledgements

We wish to thank Dr A Subramanian, CMES, Ehime

University, Japan for critical reading of the manuscript The

authors express their thankfulness to the staff of the CETASD,

Hanoi University of Science and Dr H Sakai (current

affiliation; Department of Pharmacology, Yamaguchi University

Graduate School of Medicine, Japan), Dr D Imaeda (current

affiliation; IDEA Consultants, Inc.), and Ms H Mizukawa from

CMES for their help in sample collection We also acknowledge

Ms H Touma, Ms N Tsunehiro, and Dr Ogawa, staff of the

es-BANK, CMES for their support in sample management and Ms

Y Fujii, Department of Legal Medicine, Shimane University

Faculty of Medicine, Japan for her technical assistance 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) Project for Sustainable Coexistence of Human, Nature

and the Earth (FY2002), Grants-in-Aid for Scientific Research

(S) (No 20221003 and 21221004) and (A) (No 19209025) 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

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