Genetic polymorphisms in AS3MT and arsenic metabolism in residents of the Red River Delta, Vietnam

Toxicology and Applied Pharmacology 236 (2009) 131–141

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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 ev i e r. c o m / l o c a t e / y t a a p

Genetic polymorphisms in AS3MT and arsenic metabolism in residents of the Red River Delta, Vietnam Tetsuro Agusa a,b, Hisato Iwata a,⁎, Junko Fujihara b, Takashi Kunito c, Haruo Takeshita b, Tu Binh Minh a,d, Pham Thi Kim Trang d, Pham Hung Viet d, Shinsuke Tanabe a a

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, 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 9 December 2008 Revised 23 January 2009 Accepted 25 January 2009 Available online 31 January 2009 Keywords: Arsenic AS3MT Polymorphism SNP Vietnam Groundwater Sand-filtered water Human urine Human hair

a b s t r a c t To elucidate the role of genetic factors in arsenic (As) metabolism, we studied associations of single nucleotide polymorphisms (SNPs) in As (+3 oxidation state) methyltransferase (AS3MT) with the As concentrations in hair and urine, and urinary As profile in residents in the Red River Delta, Vietnam. Concentrations of total As in groundwater were 0.7–502 μg/l. Total As levels in groundwater drastically decreased by using sand filter, indicating that the filter could be effective to remove As from raw groundwater. Concentrations of inorganic As (IAs) in urine and total As in hair of males were higher than those of females. A significant positive correlation between monomethylarsonic acid (MMA)/IAs and age in females indicates that older females have higher methylation capacity from IAs to MMA. Body mass index negatively correlated with urinary As concentrations in males. Homozygote for SNPs 4602AA, 35991GG, and 37853GG, which showed strong linkage disequilibrium (LD), had higher percentage (%) of dimethylarsinic acid (DMA) in urine. SNPs 4740 and 12590 had strong LD and associated with urinary %DMA. Although SNPs 6144,12390,14215, and 35587 comprised LD cluster, homozygotes in SNPs 12390GG and 35587CC had lower DMA/MMA in urine, suggesting low methylation capacity from MMA to DMA in homo types for these SNPs. SNPs 5913 and 8973 correlated with %MMA and %DMA, respectively. Heterozygote for SNP 14458TC had higher MMA/IAs in urine than TT homozygote, indicating that the heterozygote may have stronger methylation ability of IAs. To our knowledge, this is the first study on the association of genetic factors with As metabolism in Vietnamese. © 2009 Elsevier Inc. All rights reserved.

Introduction Consumption of arsenic (As)-polluted groundwater has adversely affected human health in certain areas of the world (Mandal and Suzuki, 2002; Nordstrom, 2002; Smedley and Kinniburgh, 2002). Recently, Berg et al. (2001) and Agusa et al. (2004, 2005, 2006, 2007, 2009) reported elevated As contamination (up to 3150 μg/l) in groundwater of the Red River Delta in Northern Vietnam; many groundwater samples contained As over the WHO drinking water guideline (10 μg/l) (WHO, 2004). It is known that As exposure causes lung and skin cancers and also birth defects (WHO, 2004). There seems to be a wide variation in the susceptibility to As toxicity among individuals and populations, which is probably related to genetic factors in metabolism of As (Vahter, 2002). It has been generally accepted that inorganic As is oxidatively methylated in the body (Challenger, 1945; Cullen and Reimer, 1989), but recently reductive methylation pathway has also been proposed ⁎ Corresponding author. Fax: +81 89 927 8172. E-mail address: [email protected] (H. Iwata). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.01.015

(Hayakawa et al., 2005; Naranmandura et al., 2006). In either case, methylation is a critical metabolic pathway for As biotransformation, since the toxicity of organic As is generally lower than that of inorganic forms, and also the methylated As would be readily excreted into the urine. In these processes, As (+ 3 oxidation state) methyltransferase (AS3MT), an S-adenosyl- L -methionine-dependent enzyme, catalyzes the methylation of arsenite (AsIII) and monomethyl As (Lin et al., 2002; Wood et al., 2006). It is known that human AS3MT gene is approximately 32-kb long and is composed of 11 exons (Wood et al., 2006). It has been reported that there are some single nucleotide polymorphisms (SNPs) in human AS3MT (Wood et al., 2006). Recombinant 173Ala N Trp (Ala to Trp substitution at amino acid base 173), 287Met N Thr, and 306Thr N Ile variants in AS3MT significantly altered levels of the enzyme activity and immunoreactive protein (Wood et al., 2006). 287Met N Thr heterozygote was linked with increased percentage of monomethylarsonic acid (MMA) in urine of central European population (Lindberg et al., 2007) and miners in Chile (Hernandez et al., 2008). For SNPs in intron of AS3MT, Meza et al. (2005) reported association between intronic SNPs 7395G NA,

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12390G N C, and 35587T N C in AS3MT, and urinary dimethylarsinic acid (DMA)/MMA values in the Mexican children. Three intronic polymorphisms in AS3MT (SNPs 12390G N C, 14215C N T, and 35991G NA) were found to be associated with a lower percentage of MMA and a higher percentage of DMA in urine of Argentina (Schläwicke Engström et al., 2007). Hence, SNPs in AS3MT may be responsible for inter-individual variation in the As metabolism. Given these results, information on the genotyping of polymorphism in AS3MT may help in understanding genetic susceptibility to As toxicity. However, data on distribution of AS3MT polymorphisms and their relations to As methylation ability are limited, especially among Asian populations (Fujihara et al., 2007). To understand the importance of certain biological and environmental factors in inter-individual variation in As metabolism, we initially investigated the effects of sex, age, body mass index (BMI), occupation, residential years, and alcohol and smoking habits on the As metabolites in urine of residents from the Red River Delta, Vietnam. Moreover, to comprehend whether or not the genetic factor can affect As metabolism, we studied the relationships between 13 SNPs in AS3MT and urinary metabolite pattern of As. Materials and methods Sampling locations and collected samples. Groundwater samples (n = 28) were randomly collected at each home in rural areas of Hoa Hau (HH) and Liem Thuan (LT) in Ha Nam Province located in the Red River Delta, Vietnam during March (dry season), 2006. There are no significant anthropogenic As pollution sources such as industrial sites and mining regions in both locations. Because some houses equipped a sand filter system for the well, filtrated groundwater samples (n = 19) were also collected along with unfiltered water samples from both locations. Human hair (n = 99), urine (n = 100), and blood (n = 100) were correspondingly collected from the residents of each house equipped with the tube well in an ethical manner. All participants were randomly selected without an arbitrary manner. We obtained the informed consent from all the subjects. The study was approved by the Ethical Committee of Ehime University, Japan. For donors who participated in this study, information on age (mean, 35.8 years; range, 11–70 years), sex (male, n = 44; female, n = 56), height (mean, 153 cm; range, 121–173 cm), weight (mean, 46 kg; range, 22–67 kg), body mass index (BMI; mean, 19.6; range, 12.1–30.0), occupation (farmer, n = 32; farmer with weaver, n = 4; weaver, n = 32; student, n = 29; retired worker, n = 2; bricklayer, n = 1), residential years (mean, 32 years; range, 3–65 years), and alcohol (yes, n = 24; no, n = 76) and smoking habits (yes, n = 20; no, n = 80) were obtained. 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 Filtered water No. Subjects No. No. of male/female Age (years)a Residential time (years)a Height (cm)a Weight (kg)a No. of smokers/non smokers No. of drinkers/non drinkers BMIa,b a b

Hoa Hau

Liem Thuan

15 9 (5.5–13) 14 (8–16)

13 6 (1–16) 15 (12–24)

10

9

51 22/29 37 (11–60) 33 (3–60) 156 (137–173) 48 (27–66) 14/37 14/37 20 (14–26)

49 22/27 34 (11–70) 31 (6–65) 150 (121–169) 44 (22–67) 6/43 10/39 19 (12–29)

Arithmetic mean and range. Body mass index (weight (kg)/height (m)2).

All samples were preserved in the Environmental Specimen Bank (es-BANK), Center for Marine Environmental Studies (CMES), Ehime University, Japan at − 25 °C (Tanabe, 2006) until chemical analysis and genotyping were conducted. The details of wells and subjects are shown in Table 1. Analyses of total As and As compounds. Water sample was acidified with nitric acid for total As (TAs) analysis. Human hair sample washed by sonication with 0.3% polyoxyethylene lauryl ether was dried for 12 h at 80 °C (Agusa et al., 2006). The dried hair sample was digested by nitric acid with a microwave oven (Agusa et al., 2006). Thawed urine was filtrated with a syringe filter (Millex-HV, 0.45 μm syringedriven filter unit, Millipore) and then diluted by Milli-Q water. Concentrations of TAs in groundwater and human hair were measured with an inductively coupled plasma-mass spectrometer (ICP-MS; HP-4500, Hewlett-Packard, Avondale, PA, USA). Yttrium was used as an internal standard for ICP-MS measurement. Six arsenic compounds including arsenocholine (AC), arsenobetaine (AB), DMA, MMA, AsIII, and arsenate (AsV) were determined in urine samples with a high performance liquid chromatograph (HPLC; LC10A Series, Shimadzu, Kyoto, Japan) coupled with ICP-MS using an anion exchange column (Shodex Asahipak ES-502N 7C) (Mandal et al., 2001; Agusa et al., 2009). The column was equilibrated with a mobile phase (15 mM citric acid, pH 2.0 with nitric acid) at a flow rate of 1.0 ml/min at 30 °C for more than 2 h before analysis. The injection volume was 50 μl. Rhodium and Rb were measured as internal standards for AB and other arsenicals, respectively. Sum of all As compounds, AsIII + AsV, and AsIII + AsV + MMA + DMA detected in urine are represented as SAs, IAs, and IMDAs, respectively. Percentages of AB, AsIII, AsV, MMA, DMA, IAs, and IMDAs to SAs in human urine were denoted as %AB, %AsIII, %AsV, %MMA, %DMA, %IAs, and %IMDAs, respectively. Urinary creatinine was measured by SRL, Inc. (Tokyo, Japan), and concentrations of As compounds in urine were expressed as μg As/g on a creatinine basis. Detection limits of TAs in water and hair, and As compounds (AC, AB, AsIII, AsV, MMA, and DMA) in urine were 0.1 μg/l, 0.01 μg/g dry wt, and 1 μg/g creatinine, respectively in our methods. To confirm the accuracy of our analytical methods, certified reference materials, SLRS-4 River Water from the National Research Council Canada (NRCC) and NIES No.13 Human Hair and NIES No.18 Human Urine provided by the National Institute for Environmental Studies (NIES), Japan were analyzed for TAs and As compounds (AB and DMA), respectively. Results of TAs in the water and hair and AB and DMA in the urine were in very good agreement with the certified values, and the recoveries were in the range of 90–106%. The analytical precision for these samples (n = 3) were within 4%. In addition, we have participated in an inter-calibration exercise organized by the Swiss Federal Institute of Aquatic Science and Technology (Eawag) in the frame of the ongoing cooperation of Vietnam and Switzerland in As related surveys and researches for analytical quality assurance and control. Genotyping of polymorphism in AS3MT. Genotyping of SNPs in AS3MT was conducted by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique (Fujihara et al. 2007). First, genomic DNA was extracted from blood sample using QIAamp DNA mini kit (Qiagen, Hilden, Germany), and genotyped for 13 SNPs in AS3MT including 4602A NG (A to G substitution at nucleotide base 4602), 4740T N C, 5913T N C, 6144A NT, 7395G NA, 8979T NA, 12390G N C, 12590T N C, 14215C N T, 14458T N C (287Met N Thr (Met to Thr substitution at amino acid base)), 35587T N C, 35991G NA, and 37853G NA. Primers for AS3MT were designed based on the DDBJ Sequence Database under accession no. AY817668 (Table 2). The mismatched PCR method (Kumar and Dunn, 1989) was employed for identifying a new restriction enzyme site for the detection of SNPs. One microgram of DNA was subjected to PCR amplification in 10 μl

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Table 2 Information on primer sequences, annealing temperatures, restriction enzymes, and fragment sizes of the amplified products used for PCR-RFLP SNP IDa

rs numberb

Functional region

Nucleotide change

Primer sequencec

Annealing temp (°C)

Restriction enzyme

Fragment size (bp)

4602

rs7085104

5′ upstream region

ANG

F: 5′-CGAAGAAACTTGTGGGCCAGA-3′ R: 5′-TCGCTCCACTGCGATTTTCAC-3′

60

MspI

5′ upstream region

TNC

F: 5′-CGAAGAAACTTGTGGGCCAGA-3′ R: 5′-CTGATTTAAATGAACACTCAC(C N G)T-3′

56.5

MslI

rs4917986

Intron 3

TNC

F: 5′-GGTCACTAGGGAATTAACCCG-3′ R: 5′-TGGCTATGTTGACCAAGCTGG-3′

61

BglI

6144

rs17878846

Intron 3

A NT

F: 5′-GGTCACTAGGGAATTAACCCG-3′ R: 5′-GGTTCCAACTAATCACCCACG-3′

61

XbaI

7395

rs12767543

Intron 3

G NA

F: 5′-CGCCTATGGGACAGAAACCTT-3′ R: 5′-CTAAGGGACAGAGT(G N C)AGACTC-3′

55

AlwNI

8979

rs7920657

Intron 5

T NA

F: 5′-AGAGTGCAGTGGCCCAATGTC-3′ R: 5′-TGAGCACAGTGCCTCACACCT-3′

63

NlaIII

12390

rs3740393

Intron 6

GNC

F: 5′-GTTCCCCTATTCCTTTC(T NA)TTG-3` R: 5′-AACCTTGGCCTCATGGCCTAA-3′

51

MslI

12590

rs3740392

Intron 7

TNC

F: 5′-GTTTCAGCATGGTGGGGAGTT-3′ R: 5′-CTG(G N C)CTATTAGC-3′

51

BslI

14215

rs3740390

Intron 8

CNT

F: 5′-CTGTACAATGGTAACCCCCCA-3′ R: 5′-GCAAGGGCAAGAGCAGAAAGA-3′

63

Hpyl88I

14458

rs11191439

Exon 9

TNC

F: 5′-GTGCTGGAGATGAACCGTGAA-3′ R: 5′-GCAAGGGCAAGAGCAGAAAGA-3′

59

HpyCH4IV

35587

rs11191453

Intron 10

TNC

F: 5′-CAGCAGTCTTGTCTTTTAAAT(ANT)AA-3′ R: 5′-CCTCTTTGGAACTGAGATACGG-3′

58

AseI

35991

rs10748835

Intron 10

G NA

F: 5′-CACGTGCAAATGCAACCCCA-3′ R: 5′-GTTTGATTTAGGTTGAC(T N G)T(A N G)CA-3′

51

ApaLI

37853

rs11191459

3′ downstream region

G NA

F: 5′-CATGGTGAGACCCCCATCTCT-3′ R: 5′-CCTGATGATAATGACC-3′

60

MspI

AA: 261 AG: 261, 200, 61 GG: 200, 61 TT: 224 TC: 224, 205, 19 CC: 205, 19 TT: 251 TC: 251, 151, 100 CC: 151, 100 AA: 415 AT: 415, 376, 39 TT: 376, 39 GG: 155 GA: 155, 138, 17 AA: 138, 17 TT: 160, 94 TA: 254, 160, 94 AA: 254 GG: 286 GC: 286, 265, 21 CC: 265, 21 TT: 164 TC: 164, 157, 7 CC: 157, 7 CC: 320, 81 CT: 401, 320, 81 TT: 401 TT: 233 TC: 233, 154, 79 CC: 154, 79 TT: 194, 21 TC: 215, 194, 21 CC: 215 GG: 205, 22 GA: 227, 205, 22 AA: 227 GG: 415, 42 GA: 457, 415, 42 AA: 457

4740

rs12416687

5913

a b c

Amino acid change

Met N Thr

SNP ID indicates the SNP identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1. Rs numbers were cited from NCBI SNP Database (http://www.ncbi.nlm.nih.gov/projects/SNP/). N N N in parenthesis indicates substitution of nucleotide for constriction of mismatched nucleotide.

reaction mixture containing GoTaq® Green Master Mix (Promega, Madison WI, USA) and each primer pair corresponding to each SNP. Information on primer sequence, annealing temperature, restriction enzyme, and PCR product size is listed in Table 2. The PCR product was digested with restriction enzyme and then was subjected to electrophoresis on 8% polyacrylamide gel. Statistical analyses. All statistical analyses were performed with StatView (version 5.0, SAS® Institute, Cary, NC, USA) and SPSS (version 12, SPSS, Chicago, IL, USA). One half of the value of the respective limit of detection was substituted for those values below the limit of detection and used in statistical analysis. All data were tested for goodness of fit to a normal distribution with Kolmogorov–Smirnov's one sample test. Because some variables were not normally distributed, log transformation was conducted for parametric analyses. Outlier (As concentration of 2120 μg/l in groundwater) was checked by Thompson test. Relationships among concentrations of As in water, hair, and urine, composition of As compounds in urine, age, residential period, and BMI were examined by Pearson's correlation coefficient test. Student's t-test was used to detect influences of regions, sexes, and smoking and drinking habits on As levels in water, hair and urine, and urinary metabolites. Differences in As concentrations between genetic polymorphisms in AS3MT were checked by Tukey–Kramer method, along with one-factor ANOVA. Relationships between genetic polymorphisms in AS3MT, and hair and

urinary As concentrations and As compositions were also examined by multiple regression analysis including sex, age, and BMI as independent variables. The residual values were obtained from the difference between the actual value and the predicted value from the regressions. These residual values reflect the variation after the effects of the regression variables have been removed. Linkage disequilibrium (LD) and haplotype of SNPs in AS3MT were calculated using Haploview (version 4.0, Day Lab at the Broad Institute Cambridge, MA, USA). A p value of less than 0.05 was considered to indicate statistical significance. Geometric mean value was represented as GM in this study. Results and discussion Concentration of total As in groundwater Arsenic was detected in all groundwater samples and the levels were 0.7–2120 μg/l. One groundwater sample with the highest concentration of TAs (2120 μg/l) was removed for further statistics, because this was considered to be an outlier (p b 0.001). The extraordinary high As level of the outlier might be due to the presence of large amount of particulate matters found in this sample. In the data set without this outlier, the range was 0.7–502 μg/l (Table 3). Arsenic concentration in groundwater from HH (GM, 368 μg/l) was significantly higher (p b 0.001) than that from LT (GM, 1.4 μg/l)

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Table 3 Concentrations (geometric mean and range) of total As and As compounds in water (μg/l), human hair (μg/g dry wt) and urine (μg/g creatinine) from Hoa Hau and Liem Thuan in Vietnam Location Groundwater n TAs⁎ Filtered water n TAs⁎ Drinking water n TAs⁎ Human hair n TAs⁎ Human urine n AC AB DMA MMA AsIII AsV SAs IAs IMDAs

Hoa Hau

Liem Thuan

15 368 (163–502, and 2120 (an outlier))

13 1.4 (0.7–6.8)

10 18.9 (3.2–143)

9 2.0 (1.0–4.9)

15 50.1 (3.2–486)

13 1.7 (0.9–4.9)

50 0.351 (0.028–2.94)

49 0.232 (0.068–0.690)

51 b1.0 17.7 (2.5–72.6) 50.5 (22.5–268) 9.3 (3.5–23.9) 6.9 (b 1.0–32.2) 1.5 (b1.0–12.7) 92.6 (45.2–365) 9.3 (3.1–38.2) 70.5 (33.6–320)

49 b 1.0 14.5 (2.1–232) 56.4 (20.2–132) 9.3 (3.8–23.1) 7.1 (b 1.0–26.6) 1.7 (b 1.0–19.1) 97.9 (38.6–397) 10.2 (4.0–28.6) 77.3 (33.0–176)

TAs; total As. SAs; sum of all As compounds. IAs; sum of AsIII + AsV. IMDAs; sum of IAs + MMAV + DMAV. Drinking water; In a house equipped with sand filter, filtered water instead of raw groundwater is assumed to be consumed. ⁎ Significant difference (p b 0.001) between locations.

(Table 3). Furthermore, all the samples (n = 15) from HH contained TAs concentrations (range, 163–502 μg/l) exceeding 10 μg/l of WHO drinking water guideline (WHO, 2004). This result suggests that the groundwater in HH is heavily contaminated by As and the health of this population is at risk. On the contrary, As concentrations in all groundwater samples from LT were less than 10 μg/l. Total As concentration in groundwater was not related to depth and usage history of the wells. Efficiency of sand filter for As removal Concentration ranges of TAs in sand-filtered groundwater were 3.2–143 μg/l in HH and 1.0–4.9 μg/l in LT (Table 3). In the filtered water, regional difference in TAs concentration was observed

(p b 0.001). In HH, concentration of TAs in filtered water (GM, 18.9 μg/l) was significantly low (p b 0.001) compared with the raw groundwater (GM, 368 μg/l), and GM of the removal efficiency of TAs from raw groundwater was estimated to be 93% (Fig. 1). Berg et al. (2006) also reported that about 80% of As was removed from groundwater by sand filtration in Vietnam. Therefore, the simple sand filter system can be useful to remove TAs from contaminated groundwater in these regions. However, several filtered water samples in HH still contained TAs concentrations over 10 μg/l of the WHO guideline value (WHO, 2004) (Fig. 1), indicating that sand filtration is not enough to remove excessive As and the water is not suitable for drinking in some cases. Concentration of total As in human hair Human hair could be a useful indicator of contamination status of trace elements including As, because it is easy for non-invasive collection, transportation, and preservation (Matsubara and Machida, 1985). Total As concentrations in hair of residents from HH and LT were in the range of 0.028–2.94 μg/g dry wt. (Table 3). Similar to the results of groundwater, TAs level in human hair of HH (GM, 0.351 μg/g dry wt) was significantly higher (p b 0.001) than that of LT (GM, 0.232 μg/g dry wt). Total As concentrations in hair of three individuals (1.00 μg/g dry wt, 2.67 μg/g dry wt, and 2.94 μg/g dry wt) from HH exceeded the level of 1 μg/g dry wt which may be a level that can induce skin lesion (Arnold et al., 1990), suggesting potential high risk for some residents in this area. Concentration and composition of As compounds in human urine It is generally known that ingested inorganic As is methylated to MMA, followed by DMA and then they are excreted into the urine in humans (Styblo et al., 2002). Therefore, As speciation is important for assessing exposure and metabolic capacity of As in humans. Concentrations of As species in human urine from the HH and LT are shown in Table 3. Concentration ranges of SAs and IMDAs were 38.6–397 μg/g creatinine and 33.0–320 μg/g creatinine, respectively. There was no significant difference in concentrations of urinary SAs between HH (GM, 92.6 μg/g creatinine) and LT (GM, 97.9 μg/g creatinine), which was inconsistent with the results of groundwater and human hair. The reason is discussed in the next section. Similar composition of urinary As compounds was found in the residents of both HH and LT, showing DMA as the predominant form and inorganic arsenicals as minor species. Arsenobetaine, which may be mainly derived from consumption of seafood, was also detected in the urine as the second abundant As species. In the present study, AC was not detected in any of the urine samples.

Fig. 1. Concentrations of TAs in raw and sand-filtrated groundwater from Hoa Hau and Liem Thuan in Vietnam.

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Relationships among concentration of As in drinking water, hair and urine To understand whether subjects in these areas are exposed to As mainly through the consumption of groundwater, relationships

Fig. 3. Comparison of (a) %IAs and (b) DMA/MMA ratio in urine between females and males from Hoa Hau and Liem Thuan in Vietnam. Bar and plots indicate arithmetic mean and individual values, respectively.

between As concentrations in water, and in hair and urine were examined. Since it is expected that residents of the house equipped with the sand filter system drink the filtered groundwater, concentration of As in filtered water was used to evaluate As exposure status for these subjects. A significant positive correlation was found between TAs concentrations of drinking water and hair in residents from HH and LT (p b 0.001; Fig. 2a). On the contrary, there were no positive correlations between concentrations of TAs in water and any of the As compounds in human urine of all the residents (the result of IMDAs is only shown in Fig. 2b). Hence, it seems that uptake of As from other source(s) such as food might affect the urinary concentration especially for the LT residents. However, since food habit was almost similar between locations but TAs levels in human hair were positively correlated with those in drinking water, peoples in HH may be not recently exposed to As from the water. Hair As levels were positively correlated with concentrations of urinary IMDAs (p b 0.001; Fig. 2c) and SAs (p = 0.002). Similar relationship was obtained in previous investigations in the population from Cambodia (Kubota et al., 2006) and Finland (Kurttio et al., 1998). Since hair can be a good indicator of past As exposure status, while As level in urine can represent recent exposure of As (Mandal and Suzuki, 2002), these results indicate that the residents in HH and LT may have been chronically exposed to As from groundwater. Fig. 2. Relationships between (a) concentrations of TAs in drinking water and human hair, (b) concentrations of TAs in drinking water and IMDAs in human urine, and (c) concentrations of TAs in human hair and IMDAs in human urine from Hoa Hau and Liem Thuan in Vietnam. Solid line in each figure represents the regression line ((a) y = 0.216x0.123, R2 = 0.155, p b 0.001; (c) y = 0.014x0.691, R2 = 0.203, p b 0.001).

Potential effects of sex, age, and BMI on As concentration and metabolism Influences of age, sex, BMI, occupation, residential years, and alcohol and smoking habits on As levels and metabolites in the residents were

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investigated. Occupation, residential period, and alcohol and smoking habits were not significantly correlated with As concentrations and profiles in the residents. Concentrations of MMA (p = 0.034), AsIII (p = 0.033), and IAs (p = 0.004) in urine and TAs (p = 0.012) in hair of males were significantly higher than those of females. Also, males showed higher %MMA (p = 0.001), %AsIII (p = 0.003), %AsV (p = 0.043), and %IAs (p b 0.001, Fig. 3a) in urine compared to females. Concentration ratio of urinary DMA/MMA, which can be a useful indicator as 2nd methylation step of IAs, was significantly higher in females (p = 0.008; Fig. 3b). These results suggest that males might be highly exposed to IAs and/or might have a lower 2nd methylation capacity of As compared to females. In our previous study, sexual difference in hair As concentration was not significant for residents in Gia Lam and Thanh Tri in Hanoi, Vietnam (Agusa et al., 2006). Interestingly, urinary As concentrations in females from As-contaminated sites were higher than those in males, while opposite trend was observed in its reference site (Chiou et al., 1997; Chowdhury et al., 2003; Loffredo et al., 2003). Although, the reason still remains unclear, our result is consistent with the study in non-As contaminated sites. Watanabe et al. (2001) reported that from an As contaminated area of Bangladesh, concentrations of As in urine of females were high but severe cases of skin pathologies were remarkably found in males, and suggested that females may prevent toxic effects (ex. skin disorder) from chronic As exposure by immediately excreting of As in their body compared with males. However, further studies on the mechanism of sexual difference in As excretion are necessary. It is known that creatinine level in urine of males is generally higher than that of females because its excretion into urine is related to mass of muscle. Therefore, it is expected that sexual difference in

Fig. 5. Relationships between BMI and (a) concentration of DMA, and (b) IMDAs in human urine from Hoa Hau and Liem Thuan in Vietnam. Solid line in each figure represents the regression line for males ((a) [Log DMA] = − 0.032 × [BMI] + 2.33, R2 = 0.278, p b 0.001, (b) [Log IMDAs] = − 0.029 × [BMI] + 2.43, R2 = 0.254, p b 0.001).

Fig. 4. Relationships between age and (a) MMA/IAs, and (b) %IAs in human urine from Hoa Hau and Liem Thuan in Vietnam. Solid lines in each figure represent the regression lines for females ((a) [MMA/IAs] = 0.018 × [age] + 0.49, R2 = 0.303, p b 0.001, (b) % IAs = − 0.075 × [age] + 12, R2 = 0.108, p = 0.013).

urinary creatinine level might affect the above-mentioned results. However, no significant difference was observed for creatinine between males and females in this study. Also, even when we used data of urinary As levels and compositions without correction by creatinine, similar results were obtained. Concentration ratio of urinary MMA/IAs, an index of 1st methylation step of IAs, significantly increased with age in all subjects (p = 0.005). Greater age-dependence was observed for females (p b 0.001, Fig. 4a). Furthermore, a significant negative correlation was observed between age and %IAs in urine of females (p = 0.013, Fig. 4b). Therefore, older persons may have a higher methylation capacity from IAs to monomethylated As than young people and also this tendency is possibly pronounced in female. Several previous studies reported that children may have a higher 2nd methylation capacity compared to adults (Agusa et al., 2009; Chowdhury et al., 2003; Chung et al., 2002), but no significant correlation between age and DMA/MMA in urine was found in the present study. This difference may be due to the small sample size of children. No age-dependent variation in human hair As concentration in this study was consistent to our previous study conducted for Vietnamese (Agusa et al., 2006). Significant negative correlations between BMI as an indicator of nutritional status (Bailey and Ferro-Luzzi, 1995) and concentrations of urinary DMA (p = 0.003), MMA (p = 0.044), AsV (p = 0.008), IAs (p = 0.006), SAs (p = 0.005), and IMDAs (p = 0.002), and hair TAs (p = 0.027) were found in all the residents. These correlations except IAs in urine and TAs in hair were also observed only in males. Representative results of DMA and IMDAs are shown in Figs. 5a and b,

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respectively. These results might suggest that nutrition status was exacerbated by As exposure in the residents, especially in males. Alternatively, people with poorer nutritional status might accumulate more As in their body. Genetic polymorphisms in AS3MT Genetic factors could also be one of critical factors for As metabolism (Vahter, 2000). The present study investigated potential influence of nine SNPs (4602, 4740, 7395, 12390, 12590, 14215, 14458, 35587, and 35991) in AS3MT that may be involved in As metabolism (Meza et al., 2005; Wood et al., 2006; Schläwicke Engström et al., 2007; Lindberg et al., 2007; Hernandez et al., 2008). Also, four SNPs

Table 4 Composition of As compounds (arithmetic mean) and concentration ratios of MMA/IAs and DMA/MMA (arithmetic mean) in urine for SNPs of AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam SNP IDa 4602 AA AG GG 4740 TT TC CC 5913 TT TC CC 6144 AA TA TT 7395 GG GA AA 8979 TT TA AA 12390 GG GC CC 12590 TT CT CC 14215 CC CT TT 14458 TT TC 35587 TT TC CC 35991 GG GA AA 37853 GG GA AA

n

%AB

%DMA

%MMA

%IAs

DMA/MMA

MMA/IAs

40 22 38

16.2 y 19.6 xy 25.8 x

61 x 59.8 x 52.7 y

10.8 10.0 10.3

11.9 10.6 11.2

5.9 6.5 5.9

1.0 1.1 1.0

52 40 8

17.1 y 24.9 x 29.4 x

61.2 y 54.9 x 45.6 x

10.3 10.1 11.3

11.5 10.1 13.7

6.5 6.0 4.6

1.0 1.2 0.9

85 14 1

22.1 15.1 31.2

57.0 60.6 43.6

9.9 y 12.7 x 13.7 xy

11.0 11.6 11.5

6.4 5.1 3.2

1.0 1.3 1.2

57 36 7

20.3 21.4 27.5

57.6 58.1 52.5

10.7 9.6 10.0

11.4 10.8 9.9

5.8 6.7 6.1

1.1 1.0 1.0

38 57 5

20.4 20.8 31.9

57.4 58.2 48.0

10.8 10.0 10.2

11.3 11.0 10.0

5.7 6.5 5.1

1.1 1.0 1.1

36 46 18

18.9 y 19.0 y 31.4 x

59.7 x 58.6 x 49.6 y

10.3 10.9 8.7

11.0 11.5 10.3

6.3 5.9 6.5

1.1 1.1 0.9

59 37 4

20.5 21.8 25.6

56.9 58.2 58.5

11.0 x 9.5 xy 7.1 y

11.6 10.6 8.9

5.6 y 6.8 x 8.2 xy

1.1 1.0 0.8

43 42 15

18.2 22.5 26.3

60.9 x 56.8 xy 48.9 y

9.8 10.3 11.7

11.1 10.4 13.0

6.7 x 6.2 xy 4.6 y

1.0 1.1 1.0

57 37 6

20.9 21.3 23.3

57.0 57.8 59.2

10.8 9.8 8.3

11.3 11.1 9.2

5.7 6.6 7.4

1.1 1.0 0.9

96 4

21.1 23.0

57.5 54.7

10.2 11.9

11.1 10.4

6.2 4.8

1.0 y 1.6 x

59 28 13

21.0 18.4 28.1

56.6 60.1 55.1

10.9 9.9 8.3

11.5 11.5 8.5

5.6 y 6.9 x 6.9 xy

1.1 0.9 1.0

19 45 36

17.2 19.6 25.3

60.2 xy 60.0 x 52.7 y

10.9 10.2 10.2

11.7 10.3 11.8

5.8 6.4 6.0

1.0 1.1 0.9

26 49 25

17.5 y 18.2 y 31.0 x

60.5 x 59.6 x 50.0 y

11.0 x 10.7 xy 8.8 y

11.1 11.6 10.2

5.7 6.2 6.5

1.2 1.0 0.9

Values with same letters are not significantly different at p b 0.05. a SNP ID indicates the SNP identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1.

137

(5913, 6144, 8979, and 37853) with relatively high frequency of alleles in AS3MT (Meza et al., 2005) were identified by PCR-RFLP in the present study. Numbers of subjects for each genotype are shown in Table 4. CC homo type of 5913 was found only in one subject and there was no homo type of 14458CC in this population. Distinct LD groups were found for SNPs in AS3MT (Fig. 6); 4602– 35991 (R2 = 0.71), 4602–37853 (R2 = 0.53), 4740–12590 (R2 = 0.61), 6144–12390 (R 2 = 0.82), 6144–14215 (R 2 = 0.72), 6144–35587 (R2 = 0.62), 12390–14215 (R2 = 0.84), 12390–35587 (R2 = 0.74), 14215–35587 (R2 = 0.78), and 35991–37853 (R2 = 0.55). In this analysis, three clusters were obtained: SNPs 4602, 35991 and 37853 as cluster 1, 4740 and 12590 as cluster 2, and 6144, 12390, 14215 and 35587 as cluster 3. Among these three LD clusters, some haplotypes were identified: 8 for LD cluster 1, 4 in LD cluster 2, and 9 in LD cluster 3 (Table 5). Haplotype 1 represented the most frequent sequence in each LD cluster of AS3MT and the frequencies in LD cluster 1, 2, and 3 were 0.457, 0.629, and 0.690, respectively. The three groups of LD obtained in this study were different from previous findings reported for the populations of Mexico (Meza et al., 2005) and Argentine (Schläwicke Engström et al., 2007) (Table 6). Mexicans had strong LD among SNPs 7395, 12390, and 35587 (Meza et al., 2005). Schläwicke Engström et al. (2007) showed a LD cluster composed of SNPs 12390, 14215, and 35991 in Argentina. Remaining genotypes such as SNPs 5913, 7395, 8979, and 14458 were independent from other SNPs in this Vietnam population. Potential effects of genetic polymorphisms in AS3MT on As concentration and metabolism Results of ANOVA followed by Tukey–Kramer Test showed statistically significant associations between 10 SNPs (4602, 4740, 5913, 8979, 12390, 12590, 14458, 35587, 35991, and 37853), and As concentration and metabolite pattern in urine of Vietnamese (Table 4). On the contrary, SNPs 6144, 7395, and 14215 had no relation to any of the indicators of As exposure and metabolic capacity. Although urinary %AB, which is unlikely to be involved in As methylation, had some associations with SNPs 4602, 4640, 8979, and 37853 (p b 0.05), these associations might result from relations between these SNPs and %DMA as shown by a strong negative correlation between %DMA and %AB (r = −0.865, p b 0.001). Concentration of TAs in human hair had no dependence on genotype in AS3MT. Homo types in 4602GG, 35991AA, and 37853AA, which had strong LD with each other (cluster 1), showed significantly lower %DMA in urine compared to other genotypes in each corresponding SNP (p b 0.05) (Table 4). For SNP 37853, %MMA in AA homozygote was also lower than those in GG type (p b 0.05, Table 4). The results did not agree with previous studies. In Mexicans, SNP 4602 was not correlated with urinary As composition (Meza et al., 2005) (Table 6). Schläwicke Engström et al. (2007) revealed that AA variant homozygosis in SNP 35991 was associated with a decrease in %MMA and an increase in % DMA in urine (Table 6), suggesting consequently a higher ratio for the 2nd methylation step. To understand relationships between As metabolic capacity and haplotype, subjects were initially divided into several groups, comprising homozygote and heterozygote for AS3MT haplotype in each LD cluster (Table 7). Then, the association was assessed by excluding haplotype groups with less than four subjects to obtain sufficient statistical power. Urinary %DMA in G1-2 (4602GG/35991AA/37853AA) of haplotype group in LD cluster 1 was significantly lower than those in G1-1 (4602AG/35991GA/37853GA) and G1-3 (4602AA/35991GG/37853GG) (p b 0.05) (Fig. 7). Furthermore, this G1-2 showed lower %MMA compared with G1-4 (4602GG/ 35991AA/37853AG) (p b 0.05). Higher %DMA in urine for TT homozygote in SNP 4740 was observed in the present study (p b 0.05) (Table 4), while no similar observation was reported in Mexicans (Meza et al., 2005) (Table 6). Although concentration of IAs in urine for 4740TT was significantly

138

T. Agusa et al. / Toxicology and Applied Pharmacology 236 (2009) 131–141

Fig. 6. Linkage disequilibrium of SNPs in AS3MT of humans from Hoa Hau and Liem Thuan in Vietnam. The value shown in each diamond indicates pair wise R2 value for each SNP pair.

higher than that for 4740TC (p = 0.002), %IA was not related to the genetic polymorphism. For SNP 12590 belonging to the cluster 2 as does SNP 4740, the TT type had higher concentrations of DMA and IMDAs, %DMA, and DMA/MMA in urine than other genotypes (p b 0.05), suggesting the prompted 2nd methylation capacity in this carrier. In the LD cluster 2, concentrations of DMA, IAs, and IMD in urine of G2-1 (4740TT/12590TT) haplotype group were greater than those in G2-2 (4740TC/12590TC) (p b 0.05) (Table 7). Furthermore, significant higher %DMA in urine was observed in G2-3 (4740TT/ 12590TC) and G2-1 than in G2-4 (4740CC/12590CC) (p b 0.05) (Fig. 7 and Table 7). Higher hair As concentration was found in G2-3

compared to G2-1, indicating the difference in allele between T and C in AS3MT 12590. Although SNPs 6144, 12390, 14215, and 35587 showed strong LD and were grouped as cluster 3 in the present study, their associations with urinary As profile were classified into two different patterns; genotypes in SNPs 6144 and 14215 showed no significant correlations with As in hair and urine, while 12390GG and 35587CC had higher urinary %MMA and thus lower DMA/MMA (Table 4). Similarly, Meza et al. (2005) and Schläwicke Engström et al. (2007) have reported lower DMA/MMA in urine of 12390GG in Mexican children and Argentina women (Table 6). SNP 14215 in females of Argentine had

Table 5 Distribution of haplotype group in linkage disequilibrium (LD) cluster og AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam SNP IDsa

Total frequency

Cumulative frequency

1 2 3 4 5 6 7 8

4602G/35991A/37853A 4602A/35991G/37853G 4602G/35991A/37853G 4602A/35991A/37853G 4602G/35991G/37853G 4602A/35991A/37853A 4602G/35991G/37853A 4602A/35991G/37853A

0.457 0.368 0.087 0.025 0.025 0.016 0.012 0.011

0.457 0.824 0.912 0.937 0.962 0.977 0.989 1.000

1 2 3 4

4740T/12590T 4740C/12590C 4740T/12590C 4740C/12590T

0.629 0.269 0.091 0.011

0.629 0.898 0.989 1.000

1 2 3 4 5 6 7 8 9

6144A/12390G/14215C/35587T 6144T/12390C/14215T/35587C 6144A/12390G/14215C/35587C 6144T/12390G/14215C/35587T 6144A/12390G/14215T/35587C 6144A/12390G/14215T/35587T 6144A/12390C/14215T/35587C 6144T/12390C/14215C/35587T 6144T/12390G/14215T/35587C

0.690 0.215 0.035 0.026 0.010 0.010 0.005 0.005 0.005

0.690 0.904 0.939 0.965 0.975 0.985 0.990 0.995 1.000

Haplotype LD cluster 1 Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype LD cluster 2 Haplotype Haplotype Haplotype Haplotype LD cluster 3 Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype Haplotype a

SNP ID indicates the SNP identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1.

T. Agusa et al. / Toxicology and Applied Pharmacology 236 (2009) 131–141

139

Table 6 Comparison of significant differences in composition of As compounds and concentration ratios of MMA/IAs and DMA/MMA in urine and linkage disequilibrium for SNPsa of AS3MT among study groups in Vietnam, Mexico, and Argentine Reference

This study

Meza et al., 2005

Schläwicke Engström et al., 2007

Study group n SNP 4602 SNP 4740 SNP 6144 SNP 7395 SNP 12390

Vietnam 100 %DMA; AA NGG, AA NGG %DMA; TT N TC, CC NS NS %MMA; GG b CC DMA/MMA; GG b GC

Mexico (children) 41 NS NS

Argentine (females) 147

SNP 12590

NS

SNP 14215

%DMA; TT N CC DMA/MMA; TT N CC NS

SNP 14458 SNP 35587

MMA/IA; TT b TC DMA/MMA; TT b TC

SNP 35991

DMA/MMA; GA N AA

SNP 37853

DMA%; GG NAA, GA N AA MMA%; GG NAA

Pair of linkage disequilibrium

SNPs 4602, 35991, and 37853 SNPs 4740 and 12590 SNPs 6144, 12390, 14215, and 35587

DMA/MMA; GG bAG + AA DMA/MMA; GG b GC + CC

%DMA; GG b GC, GG b CC %MMA; GG N GC, GG N CC DMA/MMA; GG b GC, GG b CC

%DMA; CC b CT, CC b TT %MMA; CC N CT, CC N TT DMA/MMA; CC b CT, CC b TT DMA/MMA; TT b CT + CC MMA/AsIII; TT N CT + CC %DMA; GG b GA, GG b AA %MMA; GG N GA, GG NAA DMA/MMA; GG b GA, GG b AA

SNPs 7395, 12390, and 35587

SNPs 12390, 14215, and 35991

NS; not significant. a SNP ID indicates the SNP identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1.

strong LD with SNP 12390, and the association with estimated metabolic capacity of As was also similar between SNPs 14215 and 12390 (Schläwicke Engström et al., 2007). As for Vietnamese, no such a result of SNP 14215 was observed. Among the haplotype groups in this LD cluster 3, AB concentration in urine of G3-3 (6144AT/ 12390GC/14215CT/35587CC) was significantly higher than that in G3-2 (6144AT/12390GC/14215CT/35587TC) (p b 0.05) (Table 7), although the reason was not clear. %MMA in urine for 5913TT was significantly lower (p = 0.003) than that for 5913TC (Table 4). TT genotype in 8979 was associated with higher %DMA (p b 0.05). To our knowledge, this is the first finding on the association with SNPs in addition to SNP 37853.

14458TC hetero type had significantly higher MMA/IAs (p = 0.002) than TT homo type (Table 4), although the sample size was small (n = 4). SNP 14458 located at exon 8 in AS3MT corresponds to 287 at amino acid base, in which amino acid substitution occurs from Met to Thr. A previous in vitro expression study using COS-1 cell, variant type with 287MetN Thr showed significantly higher levels of enzymatic activity and immunoreactive protein than the Met/Met homo type (Wood et al., 2006). Higher %MMA in urine was reported for 287Met N Thr heterozygous carriers than the Met/Met homozygosis in general populations in Hungary, Romania, and Slovakia, (Lindberg et al., 2007), and in male workers of copper smelting plant in Chile (Hernandez et al., 2008). Although there was no significant difference, %MMA in urine for the TC

Table 7 Composition of As compounds (arithmetic mean) and concentration ratios of MMA/IAs and DMA/MMA (arithmetic mean) in urine for haplotype group of each linkage disequilibrium (LD) cluster of AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam Group LD cluster 1 G1-1 G1-2 G1-3 G1-4 LD cluster 2 G2-1 G2-2 G2-3 G2-4 G2-5 LD cluster 3 G3-1 G3-2 G3-3 G3-4

n

Haplotype groupa

SNP IDsb

%AB

%DMA

%MMA

%IAs

DMA/MMA

MMA/IAs

33 21 17 10

Haplotype 1 × 2, 3 × 8, 4 × 7, 5 × 6 Haplotype 1 × 1 Haplotype 2 × 2 Haplotype 1 × 3

4602AG/35991GA/37853GA 4602GG/35991AA/37853AA 4602AA/35991GG/37853GG 4602GG/35991AA/37853GA

18.9 y 30.5 x 16.4 y 17.5 y

60.2 50.2 60.3 55.3

x y x xy

10.0 xy 9.0 y 11.2 xy 12.6 x

10.8 10.3 12.1 14.5

6.6 6.4 5.5 5.0

1.0 0.9 1.1 1.0

41 31 11 8 7

Haplotype 1 × 1 Haplotype 1 × 2, 3 × 4 Haplotype 1 × 3 Haplotype 2 × 2 Haplotype 2 × 3

4740TT/12590TT 4740TC/12590TC 4740TT/12590TC 4740CC/12590CC 4740TC/12590CC

18.0 25.6 13.8 29.4 22.7

60.9 55.0 62.0 45.6 52.8

x xy x y xy

9.9 9.9 11.7 11.3 12.2

11.2 9.6 12.5 13.7 12.3

6.6 6.1 6.3 4.6 4.6

1.0 1.2 1.0 0.9 1.2

54 25 7 4

Haplotype 1 × 1 Haplotype 1 × 2, 4 × 7, 5 × 8 Haplotype 2 × 3 Haplotype 2 × 2

6144AA/12390GG/14215CC/35587TT 6144AT/12390GC/14215CT/35587TC 6144AT/12390GC/14215CT/35587CC 6144TT/12390CC/14215TT/35587CC

20.4 18.6 32.2 25.6

57.6 60.0 51.6 58.5

10.7 9.6 8.4 7.1

11.3 11.8 7.9 8.9

5.8 7.0 6.5 8.2

1.1 0.9 1.2 0.8

Values with same letters are not significantly different at p b 0.05. a Each haplotype detail is shown in Table 5. b SNP ID indicates the SNP identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1.

140

T. Agusa et al. / Toxicology and Applied Pharmacology 236 (2009) 131–141

Fig. 7. Comparison of %DMA in urine among haplotype groups in linkage distribution (LD) cluster 1 and 2 from Hoa Hau and Liem Thuan in Vietnam. Bar and plots indicate arithmetic mean and individual values, respectively.

heterozygote (mean,11.9%) showed a tendency to be higher than that for the TT homozygote (mean, 10.2%) in the present study. From these results, hetero type in 14458TC (287Met N Thr at amino acid base) may have higher methylation ability from IA to MMA. Since sex, age, and BMI were also significantly related to As accumulation and methylation capacity as shown above, the results of genetic differences may be submerged by the effects of such factors. Thus, we adjusted concentrations and compositions of As using multiple regression analysis to remove effects of these co-factors and then reconsidered the genetic association with As concentration and methylation. %DMA, AB concentration and %AB in urine of local residents were not corrected by the analysis, because no significant regression equations were obtained for these variables. The adjustment by the multiple regression analysis also provided similar results to the analysis without the correction as described before. However, significant associations between %MMA and SNP 12390, between IMDAs level and SNP 12590, and between DMA, IAs, and IMDAs concentrations and haplotype groups in LD cluster 2 disappeared after the correction. On the contrary, following new associations were obtained; higher MMA/IAs for 5913TC than that for 5913TT was observed (p = 0.007), suggesting that the SNP may be related to the 1st methylation process of As. In addition, concentration of MMA for TT homozygosis in SNP 4740 was higher than TC heterozygosis (p = 0.013) and TT homozygote in SNP 5913 was associated with increased SAs level in urine (p = 0.014). Adjusted DMA and IMDAs concentrations in urine of G1-1 (4602AG/35991GA/37853GA) were the highest among LD1 haplotype groups (p b 0.05). In haplotype groups of the LD cluster 3, significant higher DMA/MMA was found in G3-2 (6144AT/12390GC/14215CT/35587TC) than in G3-1 (6144AA/ 12390GG/14215CC/35587TT). This indicates that G3-2 haplotype group may have higher 2nd methylation capacity.

The present study identified 10 SNPs in AS3MT that may affect As methylation process in residents of the Red River Delta, Vietnam. Especially, we found that SNP 12390 in AS3MT was greatly associated with DMA/MMA ratios in human urine, which was consistent with the results in previous studies (Meza et al., 2005; Schläwicke Engström et al., 2007) (Table 6). Therefore, SNP 12390 may be a universal genotype which affects 2nd step methylation process of As. Furthermore, to our knowledge, significant relationships between SNPs 5913, 8979 and 37853, and urinary As profile were observed for the first time. Interestingly, among the 10 SNPs, only SNP 14458 is located at the exon, and other SNPs are at the intron, upstream or downstream region. Further investigation is necessary to link non-exonic polymorphisms with the function of AS3MT. Acknowledgments We are grateful to Dr. A. Subramanian, CMES, Ehime University for critical reading of the manuscript. The authors wish to thank the staff of the CETASD, Hanoi University of Science and Dr. H. Sakai, CMES, Ehime University for their help in sample collection. We also acknowledge Ms. H. Touma and Ms. N. Tsunehiro, staff of the es-BANK, CMES, Ehime University for their support in sample management. This study was mainly 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 (to M.I.) and by a Grant from Research Revolution 2002 (RR2002) Project for Sustainable Coexistence of Human, Nature and the Earth (FY2002; to H.I.). Financial assistance were also provided by 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),

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