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Toxicology and Applied Pharmacology 242 (2010) 352–362 Contents lists available at ScienceDirect Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / y t a a p Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam Tetsuro Agusa a,b, Hisato Iwata b,⁎, Junko Fujihara a, Takashi Kunito c, Haruo Takeshita a, Tu Binh Minh d, Pham Thi Kim Trang d, Pham Hung Viet d, Shinsuke Tanabe b a Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, Japan Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan d Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Vietnam National University, T3 Building, 334 Nguyen Trai Street, Thanh Xuan District, Hanoi, Vietnam b c a r t i c l e i n f o Article history: Received 14 September 2009 Revised 29 October 2009 Accepted November 2009 Available online 13 November 2009 Keywords: Arsenic Glutathione S-transferase ω1 (GSTO1) GST ω2 (GSTO2) GST π1 (GSTP1) GST μ1 (GSTM1) GST θ1 (GSTT1) Genetic polymorphism Vietnam a b s t r a c t To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST ω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1 (GSTT1) wild/null There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population GSTO1 Glu155del hetero type showed higher urinary concentration of AsV than the wild homo type Higher percentage of DMAV in urine of GSTM1 wild type was observed compared with that of the null type Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from AsV to AsIII Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population © 2009 Elsevier Inc All rights reserved Introduction Inorganic arsenic (IA) is known to be a carcinogenic chemical in human Arsenic contamination in groundwater is one of the most serious health concerns in the world (Mandal and Suzuki, 2002; Nordstrom, 2002; Smedley and Kinniburgh, 2002), especially in developing countries where several areas 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: iwatah@agr.ehime-u.ac.jp (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 (+ oxidation state) methyltransferase (AS3MT) and glutathione S-transferase ω (GSTO), are required in a variety of animals including human (Aposhian and Aposhian, 2006) GST is a phase II enzyme that can detoxify xenobiotics by catalyzing the conjugation with reduced glutathione GST superfamily includes seven classes, α, μ, ω, π, θ, σ, and ζ, and the function of GSTO is different from other members of GST superfamily (Board et al., 2000) Among GSTO isoforms, GSTO1 is involved in the reduction activities of arsenate (AsV), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) (Zakharyan and Aposhian, 1999; Zakharyan et al., 2001, 2005) GSTO2 which was recently identified by Whitbread et al (2003) could catalyze the reduction of MMAV and DMAV, but its activity of DMAV reductase was remarkably lower than that of GSTO1 (Schmuck et al., 2005) It is anticipated that there is a large variation in susceptibility to toxic effect by IA among individuals and ethnics, depending on the difference in IA metabolism (Vahter, 2000) Polymorphism(s) in the genes that are responsible for the metabolism of arsenic compounds may contribute to T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 the variability in biotransformation of IA (Loffredo et al., 2003; Vahter, 2000, 2002) Our recent findings indicate significant associations of genotypes and haplotypes in AS3MT, which catalyzes the methylation of arsenite (AsIII) and monomethylarsonous acid (MMAIII) (Lin et al., 2002; Wood et al., 2006), with arsenic methylation capacity estimated by urinary arsenical profile in a Vietnamese population (Agusa et al., 2008, 2009b) Some researchers have reported the relevance of genetic polymorphisms in GSTO1 and O2 to arsenic metabolism by in vitro assays (Tanaka-Kagawa et al., 2003; Whitbread et al., 2003; Schmuck et al., 2005) and in human studies (Marnell et al., 2003; Meza et al., 2005; Paiva et al., 2008) Furthermore, there are some reports on significant associations of single nucleotide polymorphisms (SNPs) in GST π (GSTP1) and wild/null genotypes in GST μ (GSTM) and GST θ (GSTT) with biotransformation of arsenic (Chiou et al., 1997; Kile et al., 2005; Marcos et al., 2006; Zhong et al., 2006; Lin et al., 2007; McCarty et al., 2007; Steinmaus et al., 2007) However, there is no information on the distribution of gene polymorphisms in GSTs and their relation to the arsenic metabolism in Vietnamese In the present study, we investigated whether genetic polymorphisms in the members of GST superfamily, GSTO1, GSTO2, GSTM1, GSTP1, and GSTT1, can affect arsenic metabolism in residents from the Red River Delta, Vietnam The co-influence of genetic polymorphisms in GSTs and other factors (sex, age, body mass index (BMI), arsenic level in drinking water, and AS3MT genotypes) on the accumulation and metabolism of arsenic was also examined Materials and methods Samples Detailed information on samples was presented in our previous work (Agusa et al., 2009b) Well water (n = 28), human hair (n = 99), urine (n = 100), and blood (n = 100) samples were randomly collected in March 2006 from Hoa Hau (HH) and Liem 353 Thuan (LT) in Ha Nam Province, which is located in the Red River Delta, Vietnam The informed consent was obtained from all the participants, and also this study was approved by the Ethical Committee of Ehime University, Japan Data on concentrations of total arsenic in water and human hair, and arsenic compounds in urine (Agusa et al., 2009b), and cumulative arsenic exposure level are summarized in Table Cumulative As exposure level (mg) was estimated from the As level in groundwater (mg/L), year of tube-well usage (year), annual ingestion rate of groundwater (182.5 days/year), and daily water consumption (3 L/day) All samples were kept at −25 °C in a freezer of the Environmental Specimen Bank (es-BANK) in Ehime University (Tanabe, 2006) until the following analyses Analyses of arsenic The analytical method of arsenic was described in more detail elsewhere (Agusa et al., 2009b) Total arsenic (TA) in water and human hair samples was analyzed with an inductively coupled plasma-mass spectrometer (ICP-MS; HP-4500, HewlettPackard, Avondale, PA, USA) using internal calibration method Arsenic compounds including arsenobetaine (AB), DMAV, MMAV, AsIII, and AsV in urine sample were measured with a high performance liquid chromatograph (HPLC; LC10A Series, Shimadzu, Kyoto, Japan)— ICP-MS To separate each arsenical, a polymer-based anion exchange column (Shodex Asahipak ES-502N 7C) was used with 15 mM citric acid (pH 2.0 with nitric acid) (Agusa et al., 2009b) In the present study, sum of all arsenic compounds and inorganic arsenic (AsIII + AsV) detected in urine sample are denoted as SA and IA, respectively Percentages of AB, AsIII, AsV, MMAV, DMAV and IA to SA in human urine were denoted as %AB, %AsIII, %AsV, %MMAV, %DMAV and %IA, respectively Urinary creatinine was determined at SRL, Inc (Tokyo, Japan) Concentrations of arsenic compounds in urine were expressed as μg As/g on a creatinine basis Because it is considered Table Information on water and human samples from Hoa Hau and Liem Thuan in Vietnam Location Groundwater No Used period (years)a Well depth (m)a TA (μg/l)b Filtered water No TA (μg/l)b Drinking watere No TA (μg/l)b Subjects No No of male/female Age (years)a Residential time (years)a Height (cm)a Weight (kg)a No of smoker/non-smoker No of drinker/non-drinker BMIa Cumulative arsenic exposure (mg)b Hair TA (μg/g)b Urinary SA (μg/g creatinine)b Urinary AB (%)a Urinary DMAV (%)a Urinary MMAV (%)a Urinary AsIII (%)a Urinary AsV (%)a Hoa Hau Liem Thuan p-value 15 (5.5–13) 14 (8–16) 368 (163–502, and 2120 (an outlier)) 13 (1–16) 15 (12–24) 1.4 (0.7–6.8) 0.049c N 0.05c b 0.001c 10 18.9 (3.2–143) 2.0 (1.0–4.9) b 0.001c 15 50.1 (3.2–486) 13 1.7 (0.9–4.9) b 0.001c 51 22/29 37 (11–60) 33 (3–60) 156 (137–173) 48 (27–66) 14/37 14/37 20 (14–26) 306 (17.6–12800) 0.351 (0.028–2.94) 92.6 (45.2–365) 22.7 (4.0–56.8) 55.9 (32.6–77.2) 10.6 (2.9–17.8) 8.5 (0–20.3) 2.3 (0–11.1) 49 22/27 34 (11–70) 31 (6–65) 150 (121–169) 44 (22–67) 6/43 10/39 19 (12–29) 4.8 (1.7–13.4) 0.232 (0.068–0.690) 97.9 (38.6–397) 19.6 (3.1–58.6) 59.0 (29.1–78.9) 10.0 (4.8–20.9) 8.7 (0–19.8) 2.7 (0–11.3) N 0.05d N 0.05c N 0.05c 0.002c 0.027c N 0.05d N 0.05d N 0.05c b 0.001c b 0.001c N 0.05c N 0.05c N 0.05c N 0.05c N 0.05c N 0.05c Abbreviations: TA, total arsenic; BMI, body mass index (weight (kg)/height (m)2); SA, sum of arsenic compounds; AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate a Arithmetic mean and range b Geometric mean and range c t-test d χ2 test e In a house equipped with sand filter, filtered water instead of raw groundwater is assumed to be consumed 354 Table Information on primer sequences, annealing temperatures, restriction enzymes, and fragment sizes of the amplified products and frequencies of allele and genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam Amino acid position rs numbera Functional context Nucleotide change Amino acid change Primer sequences Temp (°C) Restriction enzyme PCR method Fragment size (bp) Allele Genotype frequency (%) frequency (%) GSTO1 140 rs4925 exon c→a Ala→Asp 5′-GAACTTGATGCACCCTTGGT-3′ 5′-TGATAGCTAGGAGAAATAATTAC-3′ 60 Cac8I PCR-RFLP Ala: 0.900 Asp: 0.100 GSTO1 155 rs56204475 exon agg→del Glu→del GSTO1 208 rs11509438 exon g→a Glu→Lys 5′-GCTAGGAGAAATAATTACCTCTAGC-3′ 60 5′-GAATTTACCAAGCTAGAGGAGGT-3′ 5′-GACCAAGCCAGCATTTTAGG-3′ 5′-GCAGGACAGCTTTCTGCTTT-3′ 5′-GACCTAGCTCACACCTTTCAT-3′ 37 5′-CAAAGCGCTTGGCTGTTGATGTC-3′ Ala/Ala: 68, 186 Ala/Asp: 68, 186, 254 Asp/Asp: 254 Glu/Glu: 200, 472 Glu/del: 200, 315, 472 del/del: 315, 472 Glu: 1.000 Lys: GSTO1 217 rs15032 exon c→a Thr→Asn 5′-GACCTAGCTCACACCTTTCAT-3′ 5′-CAAAGCGCTTGGCTGTTGATGTC-3′ GSTO1 236 rs11509439 exon c→t Ala→Val GSTO2 142 rs156697 exon a→g GSTP1 105 rs1695 exon a→g Glu/Glu: 154, 266 Glu/Lys: 154, 266, 420 Lys/Lys: 420 Thr/Thr: 335 Thr/Asn: 114, 221, 335 Asn/Asn: 114, 221 Ala/Ala: 116, 192 Ala/Asp: 116, 192, 308 Asp/Asp: 308 Asn/Asn: 185 Asn/Asp: 63, 122, 185 Asp/Asp: 63, 122 Ile/Ile: 176 Ile/Val: 84, 92, 176 Val/Val: 84, 92 Wild: 210 Null: Wild: 473 Null: GSTM1 GSTT1 a CTPP MboII PCR-RFLP 50 MseI PCR-RFLP 5′-CTGTGATGTCATCCTAGTTG-3′ 5′-CATGCAACCTGAACCTTGGT-3′ 50 StuI PCR-RFLP Asn→Asp 5′-AGGCAGAACAGGAACTGGAA-3′ 5′-GAGGGACCCCTTTTTGTACC-3′ 63 MBoI PCR-RFLP Ile→Val 5′-ACCCCAGGGCTCTATGGGAA-3′ 5′-TGAGGGCACAAGAAGCCCCT-3′ 55 BsmAI PCR-RFLP 5′-GAACTCCCTGAAAAGCTAAAGC-3′ 5′-GTTGGGCTCAAATATACGGTGG-3′ 5′-TTCCTTACTGGTCCTCACATCTC-3′ 5′-TCACCGGATCATGGCCAGCA-3′ 62 Rs numbers were cited from NCBI SNP Database (http://www.ncbi.nlm.nih.gov/projects/SNP/) 62 Allele specific multiplex PCR Allele specific multiplex PCR Glu: 0.955 del: 0.045 Thr: 1.000 Asn: Ala: 1.000 Asp: Asn: 0.780 Asp: 0.220 Ile: 0.845 Val: 0.155 Ala/Ala: 0.810 Ala/Asp: 0.180 Asp/Asp: 0.010 Glu/Glu: 0.910 Glu/del: 0.090 Glu/Glu: 1.000 Glu/Lys: Lys/Lys: Thr/Thr: 1.000 Thr/Asn: Asn/Asn: Ala/Ala: 1.000 Ala/Asp: Asp/Asp: Asn/Asn: 0.610 Asn/Asp: 0.340 Asp/Asp: 0.050 Ile/Ile: 0.690 Ile/Val: 0.310 Val/Val: Wild: 0.580 Null: 0.420 Wild: 0.700 Null: 0.300 T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 Gene symbol T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 that AsV, IA, and MMAV are metabolized to AsIII, MMA, and DMA, respectively, in the human body, concentration ratios of AsIII/AsV (III/ V), MMAV/IA (M/I), and DMAV/MMAV (D/M) in human urine are used as metabolic index for each arsenical Genotyping of polymorphisms in GSTO1, GSTO2, GSTP1, GSTM1, and GSTT1 Genomic DNA was extracted from the blood of 100 subjects using a QIAamp DNA mini kit (Qiagen, Chatworth, CA) Reference sequence of each GST was based on the DNA Data Bank of Japan (DDBJ); accession numbers of GSTO1, GSTO2, GSTP1, GSTM1, and GSTT1 are AY817669, AY191318, AY324387, BC024005, and AB057594, respectively DNA was subjected to PCR amplification in 10 μl reaction mixture containing GoTaq® Green Master Mix (Promega, Madison, WI, USA) and individual primer pairs corresponding to each mutation of GSTO1 Thr217Asn (threonine to asparagine substitution at amino acid base 217), GSTO1 Ala236Val, GSTP1 Ile105Val, GSTM1 wild/null, and GSTT1 wild/null For the detection of GSTO1 Glu155del and Glu208Lys, the amplification was conducted using genomic DNA, PCR buffer solution (15 mM Tris–HCl, 50 mM KCl, pH 8.0), 1.5 mM MgCl2, 0.5 μM of each primer, 200 μM dNTP, and 1.25 U Taq polymerase (AmpliTaq Gold, Applied Biosystems, CA, USA) (Fujihara et al., 2007) A PCR mixture consisting of PCR buffer, 1.5 mM MgCl2, 0.4 μM of each primer, 250 μM dNTP, and U Takara EX Taq DNA polymerase (Takara, Kyoto, Japan) was used for genotyping of GSTO1 Ala140Asp and GSTO2 Asn142Asp (Takeshita et al., 2009) Genetic polymorphisms of GSTO1 Ala140Asp, Glu208Lys, Thr217Asn, and Ala236Val, GSTO2 Asn142Asp, and GSTP1 Ile105Val were detected by PCR restriction fragment length polymorphism (PCR-RFLP) using restriction enzymes Genetic polymorphisms in GSTT1 and M1 (wild or null) were identified by allele specific multiplex PCR including β-globin as a positive control (Sreeja et al., 2005) GSTO1 Glu155del was detected by applying the method of confronting two- 355 pair primers analysis (CTPP) (Fujihara et al., 2007) The PCR products, which were treated with restriction enzyme or were not treated, were separated in 8% polyacrylamide gel by electrophoresis (300 V, 15 min) and were detected by silver staining The genotyping was carried out in duplicate The representativeness of nucleotide sequences for each genotype was confirmed with a Genetic Analyzer (model 310, Applied Biosystems Foster, CA, USA) Information on primers, annealing temperature, restricted enzyme, and fragment size is presented in Table 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 Frequencies of genotypes of GST superfamily in the Vietnamese and the HapMap populations (http://www.hapmap.org/index.html.ja) NA means no available data VN: Vietnamese in this study, CHB (H): Han Chinese in Beijing, China, CHD (D): Chinese in Metropolitan Denver, Colorado, JPT (J): Japanese in Tokyo, Japan, GIH (G): Gujarati Indians in Houston, Texas, MEX (M): Mexican ancestry in Los Angeles, California, CEU (C): Utah residents with Northern and Western European ancestry from the CEPH collection, TSI (T): Toscans in Italy, ASW (A): African ancestry in Southwest USA, LWK (L): Luhya in Webuye, Kenya, MKK (K): Maasai in Kinyawa, Kenya, YRI (Y): Yoruba in Ibadan, Nigeria 356 Table Concentrations (geometric mean and range) of arsenic compounds in urine and total arsenic in hair for each genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam GSTO1 Ala140Asp Ala/Ala Ala/Asp Asp/Asp Ala/Asp + Asp/Asp GSTO1 Glu155del Glu/Glu Glu/del GSTO2 Asn142Asp Asn/Asn Asn/Asp Asp/Asp Asn/Asp + Asp/Asp GSTP1 Ile105Val Ile/Ile Ile/Val GSTM1 Wild Null GSTT1 Wild Null n Urine Hair V V III AB DMA 81 18 19 15.2 (2.1–232) 20.6 (4.9–71.9) 18.9 20.5 (4.9–71.9) 52.6 (22.5–268) 57.0 (20.2–121) 46.5 56.4 (20.2–121) 9.3 (3.5–23.1) 9.0 (4.3–23.9) 11.1 9.1 (4.3–23.9) 7.0 (b 1.0–26.6) 6.7 (b 1.0–32.2) 11.6 6.9 (b 1.0–32.2) 91 16.1 (2.1–232) 16.0 (5.3–44.2) 53.4 (20.2–268) 52.3 (34.5–81.1) 9.3 (3.5–23.9) 8.7 (5.4–12.2) 61 34 39 15.8 17.3 12.3 16.6 51.3 56.7 55.6 56.6 (22.5–268) (20.2–121) (34.5–81.1) (20.2–121) 69 31 18.1⁎ (2.9–232) 12.4⁎ (2.1–72.6) 58 42 70 30 IA SA TA 1.6 (b 1.0–19.1) 1.7 (b 1.0–12.8) b 1.0 1.6 (b 1.0–12.8) 9.6 (3.1–35.0) 10.2 (4.5–38.2) 11.6 10.3 (4.5–38.2) 93.4 (38.6–397) 104 (40.1–227) 88.0 103 (40.1–227) 0.300 (0.028–2.94) 0.229 (0.099–0.468) NA 0.229 (0.099–0.468) 7.1 (b 1.0–32.2) 6.3 (4.0–10.0) 1.7⁎ (b1.0–19.1) 0.7⁎ (b1.0–3.0) 10.1⁎ (3.1–38.2) 6.8⁎ (4.0–10.0) 96.0 (38.6–397) 86.8 (49.5–129) 0.289 (0.028–2.94) 0.256 (0.129–0.526) 8.7 (3.5–23.1) 10.4 (4.3–23.9) 8.9 (5.4–11.8) 10.2 (4.3–23.9) 7.0 7.1 6.5 7.0 (1.7–26.6) (b 1.0–32.2) (4.2–10.0) (b 1.0–32.2) 1.5 (0.5–19.1) 2.1 (b 1.0–12.8) b 1.0 (b1.0) 1.7 (b 1.0–12.8) 9.3 (3.1–35) 11.2 (4.0–38.2) 6.5 (4.2–10.0) 10.4 (4.0–38.2) 91.6 (38.6–397) 103 (57.8–227) 87.0 (49.5–117) 101 (49.5–227) 0.296 0.265 0.311 0.271 55.3 (20.2–268) 49.1 (22.5–85.9) 9.9⁎ (3.8–23.9) 8.0⁎ (3.5–17.7) 8.3⁎⁎⁎ (b 1.0–32.2) 4.7⁎⁎⁎ (b 1.0–16.3) 1.7 (b 1.0–19.1) 1.3 (b 1.0–12.8) 11.1⁎⁎⁎ (3.2–38.2) 7.2⁎⁎⁎ (3.1–21.3) 102⁎ (38.6–397) 81.9⁎ (40.1–166) 0.294 (0.028–2.94) 0.268 (0.128–0.691) 13.6⁎ (2.1–72.6) 20.2⁎ (3.8–232) 55.9 (22.5–268) 50.0 (20.2–132) 9.2 (3.5–23.9) 9.4 (3.8–23.1) 7.1 (1.1–32.2) 6.9 (b 1.0–26.6) 1.6 (b 1.0–19.1) 1.5 (b 1.0–11.7) 9.8 (3.1–38.2) 9.6 (4.1–28.6) 95.0 (45.2–365) 95.3 (38.6–397) 0.314 (0.068–2.94) 0.251 (0.028–0.691) 15.5 (2.1–232) 17.5 (2.9–78.4) 54.5 (22.5–268) 50.8 (20.2–117) 9.2 (3.5–23.9) 9.6 (4.3–21.6) 7.0 (b 1.0–32.2) 7.1 (b 1.0–16.3) 1.6 (b 1.0–19.1) 1.6 (b 1.0–13) 9.7 (3.1–38.2) 9.8 (4.5–27.7) 95.3 (38.6–397) 94.8 (55.2–225) 0.271 (0.028–2.94) 0.324 (0.099–2.67) (2.1–232) (2.9–71.9) (5.3–32.7) (2.9–71.9) MMA As As V (0.028–2.94) (0.091–2.67) (0.134–0.526) (0.091–2.67) Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); SA, sum of arsenic compounds; TA, total arsenic; NA, not available ⁎ p b 0.05 ⁎⁎⁎ p b 0.001 T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 Gene and genotype Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); III/V, AsIII/AsV; M/I, MMAV/IA; D/M, DMAV/MMAV; NC, not calculated ⁎ p b 0.05 ⁎⁎ p b 0.01 6.4 (2.0–13.1) 5.6 (3.2–10.8) 1.0 (0.4–2.6) 1.0 (0.4–2.5) 20.2 (3.1–58.6) 23.4 (4.0–51.0) 70 30 58.5 (29.1–78.9) 54.8 (34.7–77.2) 10.2 (2.9–20.9) 10.6 (5.3–17.7) 8.6 (0–20.3) 8.6 (0–16.2) 2.5 (0–11.3) 2.5 (0–9.9) 11.1 (4.1–23.5) 11.2 (4.1–21.8) 3.6 (0.1–13.3) 3.4 (0.4–11.3) 6.5 (2.4–13.1) 5.6 (2.0–10.8) 1.0 (0.4–2.6) 1.1 (0.4–2.5) 18.5⁎ (3.1–56.8) 24.9⁎ (5.6–58.6) 58 42 60.1⁎⁎ (32.6–78.9) 53.7⁎⁎ (29.1–77.2) 10.3 (2.9–20.9) 10.3 (5.1–17.8) 8.7 (0.6–20.3) 8.6 (0–17.8) 2.5 (0–11.3) 2.4 (0–11.1) 11.2 (4.1–23.5) 11.0 (4.1–23.3) 3.4 (0.1–9.5) 3.7 (0.3–13.3) 6.0 (2.0–13.1) 6.5 (3.2–11.5) 0.9⁎⁎ (0.4–2.5) 1.2⁎⁎ (0.4–2.6) 2.5 (0–11.3) 2.4 (0–10.1) 22.2 (3.2–58.6) 18.8 (3.1–56.8) 69 31 55.7⁎ (29.1–78.9) 61.2⁎ (32.6–77.2) 10.3 (4.8–20.9) 10.3 (2.9–16.8) 9.3⁎ (0 –20.3) 7.2⁎ (0–17.8) 11.8⁎ (4.2–23.5) 9.6⁎ (4.1–23.2) 4.0⁎ (0.2–13.3) 1.9⁎ (0.1–6.1) 6.3 (2.0–13.1) 5.8 (2.4–11.1) 6.3 (5.0–6.9) 5.9 (2.4–11.1) 1.0 (0.4–2.6) 1.0 (0.4–1.9) 1.5 (1.0–2.5) 1.0 (0.4–2.5) 2.4 (0–11.3) 3.0 (0–9.9) (0–2.4) 2.6 (0–9.9) GSTO1 Ala140Asp Ala/Ala Ala/Asp Asp/Asp Ala/Asp + Asp/Asp GSTO1 Glu155del Glu/Glu Glu/del GSTO2 Asn142Asp Asn/Asn Asn/Asp Asp/Asp Asn/Asp + Asp/Asp GSTP1 Ile105Val Ile/Ile Ile/Val GSTM1 Wild Null GSTT1 Wild Null (3.1–58.6) (4.0–47.3) (9.0–39.4) (4.0–47.3) 21.4 21.5 16.9 20.9 61 34 39 57.6 (29.1–78.9) 56.0 (34.9–77.4) 65.0 (43.4–72.7) 57.2 (34.9–77.4) 10.1 10.7 10.3 10.6 (2.9–17.8) (4.8–20.9) (8.6–11.3) (4.8–20.9) 8.5 8.9 7.8 8.8 (2.4–20.3) (0–19.8) (4.1–10.2) (0–19.8) 11 (4.1–23.0) 11.9 (4.1–23.5) 7.8 (4.1–10.2) 11.3 (4.1–23.5) 3.3 (0.2–13.3) 3.9 (0.1–11.3) NC 3.9 (0.1–11.3) 6.2 (2.0–13.1) 6.1 (4.6–8.8) 1.0⁎ (0.4–2.6) 1.3⁎ (0.9–2.5) 21.3 (3.1–58.6) 20.6 (9.0–39.4) 91 57.0 (29.1–78.9) 61.1 (43.4–69.8) 10.3 (2.9–20.9) 10.1 (7.7–11.7) 8.7 (0–20.3) 7.6 (4.1–10.2) 2.7 (0–11.3) 0.7 (0–3.2) 11.4⁎ (4.1–23.5) 8.3⁎ (4.1–11.8) 3.6 (0.1–13.3) 2.4 (1.9, 2.9) 6.1 (2.0–13.1) 6.7 (3.5–11.1) 4.2 6.5 (3.5–11.1) 1.1 (0.4–2.6) 0.9 (0.5–1.9) 1.0 0.9 (0.5–1.9) 20.5 (3.1–58.6) 24.2 (4.8–47.3) 21.5 24.0 (4.8–47.3) 81 18 19 57.8 (29.1–78.9) 56.1 (34.9–77 4) 52.8 55.9 (34.9–77.4) 10.5 (2.9–20.9) 9.2 (4.8–14.8) 12.6 9.4 (4.8–14.8) 8.6 (0–20.3) 8.5 (0–15.2) 13.1 8.7 (0–15.2) 2.6 (0–11.3) 2.1 (0–7.7) 2.0 (0–7.7) 11.2 (4.1–23.5) 10.6 (4.1–17.9) 13.1 10.7 (4.1–17.9) 3.2 (0.2–13.3) 4.8 (0.1–11.3) NC 4.8 (0.1–11.3) D/M M/I III/V %IA %AsV %AsIII %MMAV %DMAV %AB n Gene and genotype Table Composition (arithmetic mean and range) of As compounds and concentration ratios of MMA/IAs and DMA/MMA in urine for each genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 357 were transformed to dummy variables The multicollinearity of independent variables was assessed by calculating the variance inflation factor (VIF) Linkage disequilibrium and haplotype of SNPs in GSTO1 were assessed by Haploview (version 4.0, Day Lab at the Broad Institute Cambridge, MA, USA) P b 0.05 was considered to indicate statistical significance Results and discussion Distribution of genetic polymorphisms in GST superfamily Allele and genotype frequencies for each gene are shown in Table There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population and thus the data on these mutations were not included for further analysis No mutation homozygotes were found for GSTO1 Glu155del and GSTP1 Ile105Val All genotypes of GSTOs and GSTP1 Ile105Val followed the Hardy– Weinberg Principle (p N 0.05) Among SNPs in GSTO1, there was no significant linkage disequilibrium and haplotype Genotype frequencies for GST superfamily in this Vietnamese and other populations which are available on HapMap (http://www hapmap.org/index.html.ja) are compared (Fig 1) Proportions of GSTO1 Ala140Asp, GSTO1 Glu208Lys, GSTO2 Asn142Asp, and GSTP1 Ile105Val genotypes in the Vietnamese population were similar to those in Asian populations such as CHB (H) (Han Chinese in Beijing, China groups), CHD (D) (Chinese in Metropolitan Denver, Colorado), and JPT (J) (Japanese in Tokyo, Japan) However, even among the Asian populations, frequencies of I/I and I/V genotypes for GSTP1 Ile105Val in the Vietnamese and Chinese (CHB (H) and CHD (D)) were largely different from those in the Japanese (JPT (J)) In addition, although mutant homo types of GSTO1 Glu208Lys and GSTP1 Ile105Val were reported in other populations, no such substitution was detected in the Vietnamese There was no mutation in GSTO1 Ala236Val in the Vietnamese Similarly, low mutation frequencies were reported in other populations except for the Mexican Genotype frequencies of GSTO1 Ala140Asp, Glu155del, and Glu208Lys, and GSTO2 Asn142Asp in the Japanese and Mongolian that have been reported in our previous studies (Fujihara et al., 2007; Takeshita et al., 2009) were close to the results on Vietnamese in this study (Table 2) For GSTO1 Glu155del and Thr217Asn, GSTM1 wild/null, and GSTT1 wild/null, the genotype frequencies of the present study were compared with those in previous studies Ninety-one percent of the Vietnamese analyzed in the present study was the wild type of GSTO1 (Table 2) and the frequency was in the range (91–100%) of previous studies (Whitbread et al., 2003; Fujihara et al., 2007; Paiva et al., 2008) No mutation allele was detected for GSTO1 Thr217Asn in the present study population (Table 2) Up to date, there is no available information on GSTO1 Thr217Asn mutation in any population, although this type has been registered in NCBI SNP Database as rs11509438 Tanaka-Kagawa et al (2003) reported that GSTO1 Thr217Asn variant had lower MMAV reductase activity compared with the wild type using in vitro assay, although the relevance of this variant in arsenic metabolism is still unclear (Schmuck et al., 2005) Null type frequencies of GSTM1 and T1 in Vietnamese were 42% and 30%, respectively (Table 2) In the review article by Mo et al (2009), the prevalence rates were shown as 41.7–55.5% in Asians, 13.1–54.5% in Caucasians, 46.7% in American-Africans, and 26.9% in Africans for GSTM1 null type and 41.9–52% in Asians, 11.1–28.6% in Caucasians, 26.7% in American-Africans, and 36.6% in Africans for GSTT1 null type Compared with the results in the Asian populations, frequency of GSTT1 deletion in the Vietnamese was lower, while proportion of GSTM1 null type was within the range On the contrary, GSTT1 null type frequency in Taiwanese was 26% (Chiou et al., 1997), which was similar to that in the Vietnamese Lin et al (2007) found 358 Table Stepwise multiple regression of arsenic concentrations and compositions in urine and hair against sex, age, BMI, TA in drinking water, and polymorphisms in GST superfamily and AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam R2adj p Independent variable β P Dependent variable R2adj p Independent variable β p %AB in urine 0.240 b 0.001 AS3MT g37853a (0 = others, = a/a) AS3MT t4740c (0 = others, = t/t) Sex (0 = female, = male) 0.359 −0.235 −0.203 b 0.001 0.013 0.024 log AB in urine 0.189 b 0.001 AS3MT g37853a (0 = others, = a/a) AS3MT t35587c (0 = others, = t/c) GSTP1 Ile105Val (0 = Ile / Ile, = Ile/Val) 0.355 −0.244 −0.217 b 0.001 0.009 0.019 %DMAV in urine 0.260 b 0.001 AS3MT t4740c (0 = others, = t/t) AS3MT g37853a (0 = others, = a/a) GSTM1 (0 = wild, = null) GSTP1 Ile105Val (0 = Ile / Ile, = Ile/Val) Age 0.282 −0.232 −0.223 0.204 0.182 0.003 0.015 0.017 0.022 0.044 log DMAV in urine 0.305 b 0.001 BMI Age AS3MT g35991a (0 = others, = g/a) AS3MT t4740c (0 = others, = t/t) −0.491 0.373 0.278 0.276 b 0.001 b 0.001 0.002 0.002 %MMAV in urine 0.306 b 0.001 Sex (0 = female, = male) AS3MT g12390c (0 = others, = g/g) AS3MT t5913c (0 = others, = t/t) AS3MT a6144t (0 = others, = a/a) AS3MT g37853a (0 = others, = a/a) 0.294 0.676 −0.254 −0.512 −0.208 b 0.001 0.002 0.004 0.020 0.022 log MMAV in urine 0.165 b 0.001 AS3MT t35587c (0 = others, = t/t) AS3MT t4740c (0 = others, = t/c) Sex (0 = female, = male) GSTP1 Ile105Val (0 = Ile /Ile, = 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, = male) GSTP1 Ile105Val (0 = Ile/Ile, = Ile/Val) 0.307 −0.225 0.002 0.019 log AsIII in urine 0.183 b 0.001 GSTP1 Ile105Val (0 = Ile/Ile, = Ile/Val) Sex (0 = female, = male) AS3MT t4740c (0 = others, = t/t) −0.342 0.245 0.193 b 0.001 0.009 0.039 %AsV in urine 0.103 0.002 AS3MT g7395a (0 = others, = g/a) GSTO1 Glu155del (0 = Glu/Glu, = Glu/del) −0.299 −0.257 0.003 0.010 log AsV in urine 0.175 b 0.001 GSTO1 Glu155del (0 = Glu/Glu, = Glu/del) BMI AS3MT g7395a (0 = others, = g/a) AS3MT t5913c (0 = others, = t/t) −0.286 −0.241 −0.237 0.186 0.004 0.012 0.015 0.050 %IA in urine 0.198 b 0.001 Sex (0 = female, = male) GSTP1 Ile105Val (0 = Ile/Ile, = Ile/Val) 0.408 −0.224 b 0.001 0.015 log IA in urine 0.291 b 0.001 GSTP1 Ile105Val (0 = Ile /Ile, = Ile/Val) Sex (0 = female, = male) AS3MT t4740c (0 = others, = t/c) BMI −0.329 0.274 −0.241 −0.177 b 0.001 0.002 0.008 0.049 III/V in urine 0.186 0.001 AS3MT c14215t (0 = others, = c/c) GSTP1 Ile105Val (0 = Ile/Ile, = Ile/Val) −0.362 −0.333 0.004 0.008 log SA in urine 0.198 b 0.001 M/I in urine 0.344 b 0.001 AS3MT g35991a (0 = others, = a/a) GSTP1 Ile105Val (0 = Ile/Ile, = Ile/Val) AS3MT t5913c (0 = others, = t/t) AS3MT t14558c (0 = t/t, = t/c) Sex (0 = female, = male) Age AS3MT t4740c (0 = others, = t/t) −0.319 0.273 −0.279 0.224 −0.201 0.204 −0.205 0.001 0.002 0.002 0.013 0.017 0.020 0.022 BMI GSTP1 Ile105Val (0 = Ile /Ile, = Ile/Val) Age AS3MT t5913c (0 = others, = t/t) −0.456 −0.229 0.271 0.216 b 0.001 0.013 0.015 0.020 log TA in hair 0.282 b 0.001 BMI log TA in drinking water Age Sex (0 = female, = male) −0.367 0.353 0.313 0.200 b 0.001 b 0.001 0.003 0.024 AS3MT g12390c (0 = others, = g/g) Sex (0 = female, = male) GSTM1 (0 = wild, = null) AS3MT t5913c (0 = others, = t/t) −0.301 −0.278 −0.271 0.240 b 0.001 0.002 0.003 0.008 D/M in urine 0.243 b 0.001 Abbreviations: AB, arsenobetaine; DMAV, dimethylarsinic acid; MMAV, monomethylarsonic acid; AsIII, arsenite; AsV, arsenate; IA, inorganic arsenic (AsIII + AsV); III/V, AsIII/AsV; M/I, MMAV/IA; D/M, DMAV/MMAV; SA, sum of arsenic compounds; TA, total arsenic; BMI, body mass index (weight (kg)/height (m)2) T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 Dependent variable T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 that prevalence of GSTM1 and T1 deletion type were 71% and 35%, respectively, in Hmong in China, and 42% and 47%, respectively, in Han in China Therefore, there may be large variations in the frequencies of GSTM1 and T1 null type even in the Asian populations Potential effects of genetic polymorphisms in GST superfamily on arsenic concentration and metabolism in Vietnamese Since the hair can be a good indicator of chronic arsenic exposure status, while arsenic level and speciation in the urine can represent recent exposure and metabolism of arsenic in humans, respectively, we measured arsenic in the hair and urine to examine their relationships to genetic polymorphisms in GST isoforms Although arsenic concentration in drinking water as well as cumulative arsenic exposure level showed significant regional difference (Table 1, p b 0.001), arsenic level and compositional profile in the urine of local people were not significantly different between HH and LT (Table 1, p N 0.05) (Agusa et al., 2009b) Also, there were no significant relationships between arsenic metabolism and TA in drinking water or cumulative arsenic exposure (p N 0.05) Thus, the data of all donors were pooled for analysis of the relationship between arsenic and genotypes of GSTs Concentrations and compositions of urinary arsenicals and metabolic index for each genotype of GST superfamily are shown in Tables and Because the sample sizes of the mutation homo types of GSTO1 Ala140Asp (n = 1) and GSTO2 Asn142Asp (n = 5) were small for the statistical analysis, these mutations were included in hetero + homo type group for the further discussion Concentrations of AsV (p = 0.018) and IA (p = 0.050) in the urine of subjects with GSTO1 Glu155del hetero type were significantly lower than those with the wild type (Table 3) Furthermore, urinary %IAs was also low in hetero type of GSTO1 Glu155del (p = 0.039, Table 4) On the other hand, higher M/I value of GSTO1 Glu155del heterozygote was observed when compared with the wild type (p = 0.039, Table 4) Whitbread et al (2003) found significantly higher activities of thioltransferase and GSH conjugation in mutation of GSTO1 Glu155del than the wild type in an in vitro study Hence, it might be suggested that if methylation of IA by AS3MT occurs in the form of arsenic-glutathione as proposed by Hayakawa et al (2005), increased GSH conjugation by GSTO1 Glu155del mutation protein enhances the methylation of IAs However, since the difference in M/I value between them was not so large in the present study and also the association between GSTO1 Glu155del and first methylation index was not obvious in the multiple regression analysis as shown later (Table 5), further studies are needed to confirm the association Schmuck et al (2005) reported that GSTO1 Glu155del protein expressed in Escherichia coli exhibited higher MMAV and DMAV reductase activities than the wild type Because MMAIII and DMAIII were not analyzed in the urine sample of the present study, relationship between genetic polymorphisms in GSTO1 and methylated trivalent arsenicals remains unclear for the subjects In the case of human studies, there was no relationship between GSTO1 Glu155del and urinary arsenical in Mexican (Meza et al., 2005) and Chilean (Paiva et al., 2008) in arsenic-contaminated regions However, Marnell et al (2003) reported that only two donors with GSTO1 Glu155del substitution in Mexicans (n = 75) who were exposed to arsenic through the consumption of drinking water showed unusual urinary arsenic profile; one who has GSTO1 Glu155del and Glu208Lys substitutions had high % AsV and low %DMAV in the urine and another one who has GSTO1 Ala140Asp substitution in addition to these two substitutions displayed high %AsIII and low %DMAV in the urine compared to the rest of the study population In the present study, no such haplotype was found As for GSTM1, concentration (p = 0.018, Table 3) and composition (p = 0.028, Table 4) of AB in the null type were significantly greater than those in the wild type, although the reason was not clear Because AB can be less metabolized in human (Cullen and Reimer, 1989), the relationship for %AB might be partially due to a negative correlation between %AB and %DMAV (p b 0.001) Urinary %DMAV 359 (p = 0.009) was low in GSTM1 null compared with that in the wild type (Table 4), implying that GSTM1 null may affect %DMAV in the Vietnamese However, the result was not consistent with those in previous studies Chiou et al (1997) reported slightly increased urinary %IA for the null genotype of GSTM1 in Taiwanese In the workers occupationally exposed to arsenic in Chile (Marcos et al., 2006) and women from arsenic-contaminated region in Argentine (Steinmaus et al., 2007), GSTM1 null type had higher %MMAV than the wild type McCarty et al (2007) found no significant association between GSTM1 genotype and methylation ratios in Bangladesh people with skin lesions No relation of GSTM1 genotype to arsenic level in toenail was reported in subjects from arsenic-endemic region in Bangladesh (Kile et al., 2005), whereas arsenic concentrations in urine and hair were high in GSTM1 null carriers exposed to indoor combustion of high arsenic coal in China (Lin et al., 2007) We found that GSTP1 Ile105Val homozygote had higher concentrations of AB, MMAV, AsIII, IA and SA, and %AsIII and %IA in the urine than the heterozygote, whereas the opposite trend was observed for % DMA (p b 0.05, Tables and 4) Metabolic index of M/I in GSTP1 Ile105Val hetero type was significantly higher than that in the wild type (p = 0.002, Table 4) Urinary III/V in the heterozygote of GSTP1 Ile105Val was about half of that in the wild homozygote (p = 0.027, Table 4), suggesting that mutation of GSTP1 Ile105Val may lead to its lower AsV reductase activity Interestingly, activity of GST in erythrocyte of GSTP1 Ile105Val wild type was higher than that of the mutation type in the healthy Chinese, but AsV reduction activity was not assessed (Zhong et al., 2006) Although GSTP1 Ile105Val variant homo type showed a slightly (p = 0.085) higher %DMAV in the urine than the wild type in Chileans (Marcos et al., 2006), the GSTP1 Ile105Val variant homo type was not observed in the population of the present study There were no significant associations of GSTO1 Ala140Asp, GSTO2 Asn142Asp, and GSTT1 wild/null with concentrations and compositions of arsenic in the urine and hair (p N 0.05, Tables and 4) The result for the polymorphism of GSTO1 Ala140Asp was consistent with the results reported in several in vitro studies; activities of MMAV reductase and DMAV reductase of GSTO1 Ala140Asp mutation were comparable to those of the wild type (Tanaka-Kagawa et al., 2003; Schmuck et al., 2005), and no significant variation was detected in the activities of thioltransferase and GST conjugation between GSTO1 Ala140Asp wild and mutation types (Whitbread et al., 2003) According to the study of Mukherjee et al (2006), protein expression level of GSTO1 Ala140Asp mutation was similar to that of the wild type, although the activity was not measured In human studies, there was no association of GSTO1 Ala140Asp with arsenic metabolism in the Mexican (Meza et al., 2005), Hungarian, Romanian, and Slovakian (Lindberg et al., 2007), and Chilean (Paiva et al., 2008) populations Like GSTO1, GSTO2 has six exons that are separated within 7.5 kb nucleotide length on chromosome 10q24.3 The homology of amino acid sequences between GSTO1 and GSTO2 is 64% (Whitbread et al., 2003) Schmuck et al (2005) reported that GSTO2 has a similar capacity to reduce MMAV but a much lower activity for DMAV as compared to GSTO1 Furthermore, GSTO2 Asn142Asp polymorphism does not affect those reductase activities This result may support our results on GSTO2 Asn142Asp which showed no association with arsenic metabolism (Tables and 4) Consistent with the results in Chilean (Marcos et al., 2006), Argentina (Steinmaus et al., 2007), and Chinese (Lin et al., 2007) subjects, GSTT1 wild/null polymorphism had no relevance to urinary arsenic in Vietnamese of the present study (Tables and 4) However, there are some available data on significant relationships between arsenic concentration and metabolism and the polymorphism Chiou et al (1997) reported that in the residents exposed to arsenic through drinking water, %DMAV in urine of subjects with GSTT1 null type was higher than that in the wild type Also, the interaction between GSTT1 wild type and secondary methylation ratio might increase the risk of 360 T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 skin lesions among arsenic-exposed individuals in Bangladesh (McCarty et al., 2007) Kile et al (2005) reported higher concentration of As in the nail of GSTT1 null type carriers in Bangladesh Concentration of TA in human hair showed a significant regional difference (Table 1, p b 0.001) and was positively correlated with TA level in drinking water (p b 0.001) (Agusa et al., 2009b) and cumulative arsenic exposure level (p b 0.001) Therefore, the association of hair TA level with polymorphisms in GST superfamily was assessed by ANCOVA, having correction with concentration of TA in drinking water and cumulative arsenic exposure as covariates, but no significant results were found (p N 0.05, Table 3) Similar to our results, there were no significant associations between arsenic contents in human hair and toenail and polymorphisms of GSTM1 and T1 in the arsenic-contaminated region in Taiwan (Chiou et al., 1997) In contrast, Lin et al (2007) found high concentrations of arsenic in both hair and urine of GSTM1 null carriers who were exposed to arsenic from indoor coal combustion in Southwest Guizhou, China, while there was no significant association between GSTT1 wild/null and arsenic levels in hair and urine Potential effects of genetic polymorphisms in GST superfamily and AS3MT, sex, age, BMI, arsenic level in drinking water, and cumulative arsenic exposure level on arsenic concentration and metabolism in Vietnamese In the previous study (Agusa et al., 2009b), we investigated the influence of various factors on arsenical concentration and composition in the urine and hair in this Vietnamese population, including age, sex, BMI, occupation, residential years, alcohol and smoking habits, and TA level in drinking water, and 13 SNPs in AS3MT such as a4602g (a to g substitution at nucleotide base 4602), t4740c, t5913c, a6144t, g7395a, t8979a, g12390c, t12590c, c14215t, t14458c, t35587c, g35991a, and g37853a, and revealed significant effects of sex, age, BMI, concentration of TA in drinking water, and several SNPs in AS3MT Therefore, those factors might co-affect the results on relationships between arsenic and GST genotypes examined in this study In the previous study by Lindberg et al (2007), multiple regression analyses was conducted to assess whether the distributions of urinary arsenic metabolites were dependent on sex, age, BMI, selenium, and gene polymorphisms of GSTO1, AS3MT, and methylenetetrahydrofolate reductase (MTHFR) Here, we attempted to detect the effects of sex, age, BMI, concentration of arsenic in water, cumulative arsenic exposure status, and polymorphisms in GST superfamily and AS3MT on arsenic level and profile in the Vietnamese using a stepwise multiple regression analysis Prior to the analysis, we confirmed that there were no significant variations in sample numbers of each sex, age and BMI in each genotype of GST superfamily (p N 0.05) The multicollinearity of independent variables for multiple regression analysis was examined by calculating the VIF The result showed that there was no significant multicollinearity (all VIFs were less than 10) The result of stepwise multiple regression analysis is shown in Table This multivariate assessment showed similar results to those of univariate analysis in the present study (Tables and 4) and our previous results (Agusa et al., 2009b): associations between GSTO1 Glu155del and concentration of AsV in urine; between GSTP1 Ile105Val and concentrations of AB, MMAV, AsIII, IA and SA, %DMAV, %AsIII, %IA, III/V and M/I in urine; between GSTM1 wild/null and %DMAV in urine; between age and M/I; between sex and concentrations of MMAV, AsIII and IA, %MMAV, %AsIII, %IA and D/M in urine, and concentration of TA in hair; between BMI and concentrations of DMAV, AsV, IA and SA in urine, and TA in hair; between concentrations of TA in drinking water and hair; between AS3MT t4740c and concentrations of DMAV, MMAV and IA, %AB, and %DMAV in urine; between AS3MT t5913c and concentration of SA, %MMAV, and M/I in urine; between AS3MT g12390c and %MMAV and D/M in urine; between AS3MT t14458c and M/I in urine; between AS3MT g35991a and %DMAV in urine; and between AS3MT g37853a and concentration of AB, %AB, %DMAV, and %MMAV in urine Furthermore, several associations were newly identified by the multiple regression analysis: associations of GSTO1 Glu155del with %AsV in urine; of GSTM1 wild/null with D/M in urine; of age with DMAV level, SA level and %DMAV in urine, and with TA level in hair; of sex with %AB and M/I in urine; of AS3MT t4740c with AsIII level and M/I in urine; of AS3MT t5913c with AsV level and D/M in urine; of AS3MT a6144t with %MMAV in urine; of AS3MT g7395a with AsV level and %AsV in urine; of AS3MT c14215t with III/V in urine; of AS3MT t35587c with AB and MMAV levels in urine; and of AS3MT g35991a with M/I in urine Factors influencing metabolic capacity of arsenic were also characterized as follows: lower III/V in c/c homo type of AS3MT c14215t and Ile/Val hetero type of GSTP1 Ile105Val; lower M/I in a/a homo type of AS3MT g35991a, Ile/Ile homo type of GSTP1 Ile105Val, t/t homo type of AS3MT t5913c, t/t homo type of AS3MT t14558c, male, younger people, and t/t homo type of AS3MT t4740c; and lower D/M in g/g homo type of AS3MT g12390c, male, null type of GSTM1, and c/c + t/ c types of AS3MT t5913c Although we statistically detected co-effects of genetic polymorphisms in GST superfamily as well as sex, age, BMI, TA in drinking water and AS3MT genotypes on the arsenic concentration and metabolism, adjusted determination coefficient (R2adj) in multiple regression equation was not so high (up to 0.344 for M/I) (Table 5) Thus, it seems possible that there are other significant factors to account for the variation in arsenic level and metabolism Other SNP sites in GSTO1 (Mukherjee et al., 2006) and AS3MT (Wood et al., 2006), which are not determined in the present study, might also be involved in the variation Mukherjee et al (2006) found large variations of protein expression levels of GSTO1 Cys32Tyr and GSTO2 Val41Ile, Cys130Tyr, and L158Ile as well as those investigated in the present study (GSTO1 Ala140Asp, Glu155del, Glu105Lys, and Ala236Val, and GSTO2 Asn142Asp), indicating the possibility of significant variations in the expression levels and catalytic functions among polymorphisms in GSTO1 and GSTO2 Also, recombinant Arg173Trp and Thr306Ile variants in AS3MT significantly suppressed levels of the enzyme activity and immunoreactive protein compared to the wild type (Wood et al., 2006) However, those variant allele frequencies are quite low (up to 5%) in African-Americans, Caucasian-Americans, Han Chinese-Americans, and Mexican-Americans (Mukherjee et al., 2006; Wood et al., 2006), indicating the difficulty in confirming the relationship among the population at small sample scale Many SNPs in the intron regions of GSTO1, GSTO2 and AS3MT have been reported by Mukherjee et al (2006), Wood et al (2006), and NCBI SNP Database, and the linkage between the intronic polymorphisms and metabolism of arsenic is of interest Alternatively, other genetic polymorphisms such as MTHFR Ala222Val and Glu429Ala, which may associate with arsenic metabolism in human (Lindberg et al., 2007; Schläwicke Engström et al., 2007; Steinmaus et al., 2007), should be considered Further studies at larger scale are required to detect more rigid relationships between genetic polymorphisms and arsenic metabolism Also, several nutritional factors such as β-carotene, selenium and vitamins C and E are known to modify toxicity of arsenic (Schoen et al., 2004) and thus such factors may influence arsenic metabolism for residents in developing countries In summary, we suggest here that genotypes of GSTO1 Glu155del, GSTP1 Ile105Val, and GSTM1 wild/null affect arsenic metabolism in a Vietnamese population Interestingly, GSTP1 Ile105Val polymorphism, on which there is little information in association with arsenic metabolism, showed statistically significant and wide associations with urinary arsenic In addition to the genetic polymorphisms of GST superfamily, sex, age, BMI, TA in the drinking water, and various SNPs in AS3MT were also related to arsenic level and profile in the Vietnamese To our knowledge, this is the first comprehensive study indicating the associations between genetic polymorphisms of GSTs and arsenic metabolism in a Vietnamese population T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362 Acknowledgments We wish to thank Dr A Subramanian, CMES, Ehime University, Japan for critical reading of the manuscript The authors express thanks to the staff of the CETASD, Hanoi University of Science and Dr H Sakai, CMES (current affiliation; Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Japan) for their help in sample collection We also acknowledge Ms H Touma and Ms N Tsunehiro, staff of the esBANK, CMES for their support in sample management and Ms Y Fujii, Department of Legal Medicine, Shimane University Faculty of Medicine, Japan (current affiliation; Department of Immunology, Shimane University Faculty of Medicine, Japan) for her technical assistant This study was supported by Japan Society for the Promotion of Science (JSPS) for the cooperative research program under the Core University Program between JSPS and Vietnamese Academy of Science and Technology (VAST) Financial support was also provided by grants from Research Revolution 2002 (RR2002; to H.I.) 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their relationships to genetic polymorphisms in GST isoforms Although arsenic concentration in drinking... regression of arsenic concentrations and compositions in urine and hair against sex, age, BMI, TA in drinking water, and polymorphisms in GST superfamily and AS3MT in residents from Hoa Hau and Liem