Gene polymorphisms of glutathione S-transferase omega 1 and 2, urinary arsenic methylation profile and urothelial carcinoma.

(1)

Gene polymorphisms of glutathione S-transferase omega 1 and 2, urinary arsenic

methylation pro

ﬁle and urothelial carcinoma

Chi-Jung Chung

a,1

, Yeong-Shiau Pu

b

, Chien-Tien Su

c

, Chao-Yuan Huang

b,d

, Yu-Mei Hsueh

a,e,

⁎

a

School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan

b_{Department of Urology, National Taiwan University Hospital, Taipei, Taiwan} c

Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan

d

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University

e

Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

a b s t r a c t

a r t i c l e i n f o

Article history: Received 29 June 2010

Received in revised form 28 October 2010 Accepted 28 October 2010

Available online 21 November 2010 Keywords:

Arsenic methylation proﬁle GSTO1

GSTO2 Polymorphism Urothelial carcinoma

Genetic polymorphisms in arsenic-metabolizing enzymes may be involved in the biotransformation of inorganic arsenic and may increase the risk of developing urothelial carcinoma (UC). The present study evaluated the roles of glutathione S-transferase omega 1 (GSTO1) and GSTO2 polymorphisms in UC carcinogenesis. A hospital-based case-control study was conducted. Questionnaire information and biological specimens were collected from 149 UC cases and 251 healthy controls in a non-obvious inorganic arsenic exposure area in Taipei, Taiwan. The urinary arsenic profile was determined using high-performance liquid chromatography and hydride generator-atomic absorption spectrometry. Genotyping for GSTO1 Ala140Asp and GSTO2 Asn142Asp was conducted using polymerase chain reaction–restriction fragment length polymerase. GSTO1 Glu208Lys genotyping was performed using high-throughput matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. A significant positive association was found between total arsenic, inorganic arsenic percentage and monomethylarsonic acid percentage and UC, while dimethylarsinic acid percentage was significantly inversely associated with UC. The minor allele frequency of GSTO1 Ala140Asp, GSTO1 Glu208Lys and GSTO2 Asn142Asp was 18%, 1% and 26%, respectively. A significantly higher MMA% was found in people who carried the wild type of GSTO1 140 Ala/Ala compared to those who carried the GSTO1 140 Ala/Asp and Asp/Asp genotype (p = 0.02). The homogenous variant genotype of GSTO2 142 Asp/Asp was inversely associated with UC risk (OR = 0.17; 95% CI, 0.03 - 0.88; p = 0.03). Large-scale studies will be required to verify the association between the single nucleotide polymorphisms of arsenic-metabolism-related enzymes and UC risk.

1. Introduction

Urothelial carcinoma (UC) includes malignances of the bladder, the renal pelvis, the ureter and the urethra. Occupational exposure to aromatic amines, cigarette smoking and inorganic arsenic in drinking water are well known risk factors for the development of UC (Negri and

La, 2001). Epidemiological evidence has demonstrated that exposure to

arsenic in drinking water is associated with skin cancer, liver cancer and bladder cancer (Hsueh et al., 1997; Chiou et al., 1995). The signiﬁcant correlation between urinary arsenic proﬁle and UC was demonstrated in our previous study (Pu et al., 2007), as well as other studies (Chen et al.,

2003; Steinmaus et al., 2006). These studies showed that people with

unfavorable urinary arsenic profiles, e.g., higher total arsenic, higher inorganic arsenic percentage (InAs%), higher monomethylarsonic acid percentage (MMA%), or lower dimethylarsinic acid percentage (DMA%), had higher rates of UC than those with more favorable arsenic profiles. Arsenic-induced carcinogenesis in the human bladder may be due to the fact that the bladder is exposed to high levels of arsenic, as it is bioconcentrated in urine (Chen et al., 1988). Upon entering the human body, inorganic arsenic is enzymatically transformed to MMA, DMA, and, in some species, trimethyl arsenic (TMA) (Cohen et al., 2006). Among these species, the trivalent arsenicals, particularly the trivalent methylated arsenic metabolites, have been identified as the most toxic forms of arsenic (Styblo et al., 2000). The toxicological effects of arsenic on urothelium have been established through UROtsa cells, including inducing malignant transformation, mediating cell proliferation and gene expression by upregulation of activating protein-1, as well as disturbing other signal transduction pathways (Eblin et al., 2008; Sens

et al., 2004; Simeonova et al., 2000; Su et al., 2006).

To date, arsenic (+3 oxidation state)-methyltransferase (AS3MT), purine nucleoside phosphorylase (PNP), glutathione S-transferase omega ⁎ Corresponding author. Department of Public Health, School of Medicine, College of

Medicine, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: +886 2 27361661x6513; fax: +886 2 27384831.

E-mail address:ymhsueh@tmu.edu.tw(Y.-M. Hsueh).

1

Current address: Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.

Contents lists available atScienceDirect

Science of the Total Environment

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 / s c i to t e n v

(2)

1 (GSTO1) and GSTO2 have been proposed to be involved in the arsenic metabolism pathway (Aposhian et al., 2004; Aposhian and Aposhian,

2006; Del Razo et al., 2001; Schmuck et al., 2005). AS3MT likely catalyzes

the conversion of arsenite (iAs3+_{) methylate to MMA}5+_{and of MMA}3+

methylate to DMA5+_{using S-adenosyl-methionine (SAM) as the methyl}

donor (Drobna et al., 2004, 2005, 2006; Lin et al., 2002; Thomas et al., 2007). Furthermore, PNP, GSTO1 and GSTO2 may be involved in reducing the pentavalent arsenic species, including arsenate (iAs5+_{), MMA}5+_{, and}

DMA5+, to trivalent arsenicals (Chowdhury et al., 2006; Radabaugh et al.,

2002).

Individual genetic susceptibility may affect inorganic arsenic metabolic capability, changing the proﬁle of urinary arsenic species and, thus, the potential to develop UC. Several single nucleotide polymorphisms (SNPs) have been reported in GSTO1 and GSTO2 (Kolsch et al., 2007; Leite et al., 2007). A genetic association study evaluated six polymorphic sites in GSTO1 and GSTO2 in 100 Vietnam people, and found that people with GSTO1 Glu155del heterozygous genotype had higher urinary iAs5+_than

those with the wild homozygous genotype (Agusa et al., 2010). In addition, other studies found no associations between GSTO1 exon4 Ala140Asp (or GSTO2 Asn142Asp) gene polymorphism and urinary arsenic profile, including Chinese populations chronically exposed to arsenic in drinking water and copper mine workers occupationally exposed to arsenic (Paiva et al., 2008; Xu et al., 2009). Previous studies found conflicting results about the polymorphism of GSTO1 Ala140Asp related to the disease risks. For example, the polymorphism of GSTO1 Ala140Asp increased risks for the development of cerebrovascular atherosclerosis, hepatocellular carcinoma, cholangiocarcinoma, breast cancer (Kolsch et al., 2007; Marahatta et al., 2006) and a reduced risk for Parkinson's disease (Wahner et al., 2007). There were few studies exploring the relationship among the polymorphisms of arsenic-metabolized enzymes, urinary arsenic profile and UC risk. De Chaudhuri et al. attempted to analyze the association between these factors and arsenic-related skin lesions and found that the polymorphisms of GSTO1 Ala140Asp and GSTO2 Asn142Asp were not associated with arsenic-induced skin lesions and skin cancer in West Bengal individuals

(De Chaudhuri et al., 2008).

Therefore, this study explored whether the arsenic-metabolizing genes of GSTO1 Ala140Asp, GSTO1 Glu208Lys and GSTO2 Asn142Asp affected the urinary arsenic proﬁle and evaluated a possible association between these SNPs and the development of UC. 2. Materials and methods

2.1. Study area and participants

We conducted a hospital-based case-control study according to the protocol and recruitment strategy described previously (Pu et al., 2007). Brieﬂy, we recruited 223 UC cases and 607 healthy controls from the Medical Center, which includes National Taiwan University Hospital and Taipei Municipal Wan Fang Hospital, from September 2002 to August 2007. Informed consent forms were provided to all participants prior to questionnaire interviews and collection of biological specimens. The Research Ethics Committee of the National Taiwan University Hospital, Taipei, Taiwan, approved the study and it was consistent with the World Medical Association Declaration of Helsinki. All UC cases were diagnosed by histological conﬁrmation. Healthy controls with no prior history of cancer were matched to UC cases in terms of age (±5 years) and gender. A total of 185 UC cases and 412 healthy controls were matched.

2.2. Questionnaire interview and biological specimen collection Well-trained interviewers collected detailed information through a face-to-face interview. The context of the structured questionnaire included demographics and socioeconomic characteristics, lifestyle habits such as cigarette smoking and alcohol consumption, residential and occupational history, and personal and family histories of UC. Spot urine

samples were collected at the time of recruitment and immediately transferred to a −20 °C freezer until the analysis of urinary arsenic pro_{ﬁles. Simultaneously, blood samples were collected and frozen at} −80 °C for DNA extraction. Among study participants, the questionnaire data or biological specimens of 36 cases and 161 controls were unavailable for collection. Finally a total of 149 UC cases and 251 healthy controls were included in the present study.

2.3. Urinary arsenic proﬁles assessment

Urinary arsenic proﬁles of iAs3+_{, iAs}5+_{, MMA}5+_{and DMA}5+_were

analyzed by high-performance liquid chromatography, equipped with a hydride generator and atomic absorption spectrometer. Detailed methods for the analysis of these species have been reported in our previous study (Hsueh et al., 1998). As a quality control, freeze-dried SRM 2670 urine obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) containing 480 ± 100μg/L arsenic were analyzed along with the urine specimens of study subjects. The arsenic value in SRM 2670 was determined to be 507 ± 17_μg/L (n= 4). Recovery rates of the four arsenic species were calculated using the following formula: ([(sample spiked standard solution concentra-tion)−sample concentration] /standard solution concentration×100). The recovery rates of iAs3+_{, DMA}5+_{, MMA}5+_{, and iAs}5+_{were from 93.8}

to 102.2%, with detection limits of 0.02, 0.08, 0.05, and 0.07μg/L, respectively. In consideration of the stability of urinary arsenic proﬁles, the assay of arsenic species was performed within 6 months of the collection (Chen et al., 2002). In addition, previous studies have demonstrated that seafood may affect the levels of urinary arsenic species (Ma and Le, 1998; Francesconi and Kuehnelt, 2004); however, our previous study found that the levels of iAs3±_{, iAs}5±_{, MMA, DMA, total}

arsenic, InAs%, MMA% and DMA% were similar before and after 3 days of seafood restriction (Hsueh et al., 2002). We also found that the frequencies offish, shellfish and seaweed intake do not significantly correlate with urinary arsenic species (Hsueh et al., 2002). These results were consistent with thefindings of Lin (Lin, 1986). Therefore, it is unlikely that urinary arsenic species are confound by the consumption of seafood within 3 days in this study.

2.4. Genotyping

Genomic DNA was extracted from blood specimens using proteinase K digestion following phenol and chloroform extraction. Genotyping for SNPs in GSTO1 Glu208Lys (rs11509438) was performed using high-throughput matrix-assisted laser desorption and ionization time-of-ﬂight (MALDI-TOF) mass spectrometry (SEQUENOM MassARRAY system; Sequenom, San Diego, CA, USA). The PCR primers for GSTO1 Glu208Lys were designed using Spectro-Designer software (SEQUENOM, Inc.). The necessary sequence information for primer design was based on the GenePipe database (http://genepipe.ngc.sinica.

edu.tw/seqtool/pages/getSeq.jsp). Information on the primers is

available from the authors upon request. Briefly, uniplex polymerase chain reaction (PCR) was carried out by the forward and reverse primers. After primer extension, the purified DNA fragments were spotted onto a 384-element silicon chip and analyzed in the Bruker Biflex III MALDI-TOF SpectroREADER mass spectrometer. The resulting spectra were processed with SpectroTYPER (Sequenom). To ensure the specificity and reliability of observed polymorphisms, the results were confirmed by repeating 10% of the assays. In addition, genotyping for GSTO1 Ala140Asp (rs4925) and GSTO2 Asn142Asp (rs156697) was carried out by using the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique (Marahatta et al, 2006). In brief, the primers 5′-GAA CTT GAT GCA CCC TTG GT-3′ (forward) and 5′-TGA TAG CTA GGA GAA ATA ATT AC-3′ (backward) for GSTO1 Ala140Asp polymorphism, 5′-AGG CAG AAC AGG AAC TGG AA-3′ and 5′-GAG GGA CCC CTT TTT GTA CC-3′ for GSTO2 Asn142Asp polymor-phism were used to amplify 254 bp and 185 bp PCR products,

(3)

respectively. PCR products were obtained in a total volume of 30μL, containing an 80 ng sample DNA, 10× PCR buffer, 2.5 mM dNTP, 2μM of each primer and 2 U Taq polymerase. After initial denaturation for 5 min at 94 °C, 30 cycles were performed at 94 °C for 1 min (denaturation), followed by 60 °C for 1 min (annealing) andfinally 72 °C for 1 min (extension) forGSTO1, and for GSTO2 an additional_{final step at 72 °C for} 5 min was included in each cycle. The amplified products were visualized by electrophoresis in a 2% agarose gel. PCR products were digested with Cac8 I (18 h, at 37 °C) for GSTO1 and Mbo I (18 h, at 37 °C) for GSTO2. Genotypes were analyzed by electrophoresis on 3% agarose gels. For quality control, a random 5% of the samples were repeated with a concordance of 100%.

2.5. Statistical analysis

All data analyses were performed by using the SAS package (SAS, version 8.0, Cary, NC). The urinary total arsenic concentration was the sum of iAs3+_{, iAs}5+_{, MMA}5+_{, and DMA}5+_{and was normalized against}

urinary creatinine levels (μg/g creatinine). The relative proportion of each arsenic species (InAs (iAs3+_{+ iAs}5+_{)%, MMA% and DMA%) was}

estimated by dividing the concentration of each arsenic species by the total arsenic concentration. The frequency distributions of GSTO1 and GSTO2 polymorphisms were evaluated in controls to test the Hardy– Weinberg equilibrium. We calculated cumulative exposure of cigarette smoking (pack-years) using the information of smoking status (never, current or past) and daily numbers of cigarettes smoked. We used multivariate logistic regressions to estimate the odds ratios (OR) and 95% confidence intervals (CI) on UC risk associated with relevant variables, including urinary arsenic profiles and gene polymorphisms of GSTO1 and GSTO2. Finally, we tested the differences of urinary arsenic pro_{files among polymorphisms of GSTO1 and GSTO2 using the} Wilcoxon rank-sum test or the Kruskal–Wallis test.

3. Results

The sociodemographic characteristics of cases and controls are shown

inTable 1. Participants who had higher educational levels had a lower risk

of UC than those with lower educational levels. Subjects who had paternal ethnicity of Mainland Chinese had a lower risk of UC than those with paternal ethnicity of Fukien Taiwanese. Occasional alcohol drinkers had a significantly lower UC risk than non-drinkers and frequent drinkers. Pesticide users or participants with cumulative cigarette smokingN0 had a significantly higher 2.6-fold UC risk than non-users or those with cumulative cigarette smoking=0. Age and gender, as well as other potential confounders including coffee drinking or hair dye use, did not affect the UC risk. The median of urinary total arsenic levels in 149 patients of UC was significantly higher than 251 controls (29.78 μg/g creatinine vs.16.47μg/g creatinine; p≤0.05). A significantly elevated risk of UC was related with urinary total arsenic, or InAs%, or MMA% increment; however, a significantly reduced risk of UC was related with urinary DMA% increment after adjustments for age, gender, education, paternal ethnicity, cumulative cigarette smoking, alcohol drinking and pesticide usage (p≤0.01).

InTable 2, the distributions of GSTO1 Ala140Asp, GSTO1 Glu208Lys

and GSTO2 Asn142Asp polymorphisms wereﬁtted the Hardy–Weinberg equilibrium respectively in controls (p≥0.05). Participants who carried the homogeneous variant genotype of GSTO2 142 Asp/Asp had a signiﬁcantly protective risk of UC (OR=0.17; 95% CI, 0.03 - 0.88; p = 0.03) compared with those who carried the wild type of GSTO2 142 Asn/Asn after adjustment for age, gender and other potential confounders. However, any association was not observed between the polymorphisms of GSTO1 Ala140Asp or GSTO1 Glu208Lys and UC risk. In addition, no association was found between paternal ethnicity and genetic frequency of GSTO1 or GSTO2 (data not shown).

We compared the urinary arsenic pro_{ﬁles in different genotypes of} GSTO1 or GSTO2 among control groups (n = 251) (inTable 3). The

Table 1

Sociodemographic characteristics and urinary arsenic proﬁles for UC cases and controls.

Variables UC cases (n = 149) no. (%) Control (n = 251) no. (%) Age and gender adjusted OR (95% CI) p Age (years) (Mean ± SE) 63.24 ± 1.04 62.69 ± 0.81 1.00 (0.99–1.02) 0.67 Gender

Male 104 (69.80) 178 (70.92) 1.00

Female 45 (30.20) 73 (29.08) 0.94 (0.60–1.46) 0.77

Highest educational level

Elementary school or below 66 (44.30) 39 (15.66) 1.00&

High school 56 (37.58) 90 (36.14) 0.31 (0.18–0.54) ≤0.01

College or above 27 (18.12) 120 (48.19) 0.10 (0.05–0.19) ≤0.01 Paternal ethnicity

Fukien Taiwanese 108 (72.48) 131 (52.19) 1.00&

Hakka Taiwanese 14 (9.40) 30 (11.94) 0.57 (0.29–1.12) 0.10

Mainland Chinese 27 (18.12) 90 (35.86) 0.34 (0.20–0.58) ≤0.01

Cumulative cigarette smoking (pack-years) 18.75 ± 2.34 9.44 ± 1.14 1.02 (1.01–1.03) ≤0.01

0 74 (51.39) 162 (66.94) 1.00 N0a _{70 (48.61)} _{80 (33.06)} _{2.62 (1.55–4.41)} _≤0.01 Alcohol drinking Never 93 (62.42) 123 (49.00) 1.00 Occasional 25 (16.78) 89 (35.46) 0.37 (0.22–0.63) ≤0.01 Regular 31 (20.81) 39 (15.54) 1.02 (0.57–1.84) 0.94 Pesticide usage No 130 (87.84) 238 (94.82) 1.00 Yes 18 (12.16) 13 (5.18) 2.59 (1.23–5.48) 0.01

Urinary arsenic proﬁle Median±SE

Total arsenic (μg/g creatinine) 29.78 ± 3.97 16.47 ± 0.93 1.03 (1.02–1.05)b

≤0.01

InAs% 6.47 ± 1.08 4.24 ± 0.63 1.03 (1.01–1.06)b _≤0.01

MMA% 9.05 ± 0.95 5.76 ± 0.46 1.03 (1.01–1.06)b

0.01

DMA% 83.68 ± 1.44 88.74 ± 0.75 0.97 (0.95–0.98)b _≤0.01

SE: standard error.

a

Including people who were ever-smokers or who were smokers.

b

Multivariate ORs were adjusted for the highest educational level, paternal ethnicity, cumulative cigarette smoking, alcohol drinking and pesticide usage.

(4)

results revealed a signiﬁcantly higher MMA% in people who carried the wild type of GSTO1 140 Ala/Ala, as compared to those who carried the GSTO1 140 Ala/Asp and Asp/Asp genotype (p = 0.02). Urinary MMA% level was signiﬁcantly different among three groups of people who carried GSTO2 Asn142Asp polymorphism (p = 0.01). However, an association was not observed between other genotypes of GSTO1 Ala140Asp, GSTO1 Glu208Lys or GSTO2 Asn142Asp and the levels of urinary total arsenic or InAs% or DMA%. In addition, while there is no homogeneous variant genotype of GSTO1 208 Lys/Lys; however, people with the heterozygous genotype of GSTO1 208 Glu/Lys had slightly higher iAs3+_{% than those with the wild type of GSTO1 208}

Glu/Glu (0.05bpb0.1; data not shown). 4. Discussion

To our knowledge, the present study is thefirst to evaluate the impact of GSTO1 and GSTO2 gene polymorphisms on UC susceptibility in a non-obvious inorganic arsenic exposure area in Taiwan. The GSTO2 142 Asp/ Asp genotype was found to be significantly protective against UC risk. Furthermore, a significantly higher MMA% was observed in people who carried the wild type of GSTO1 140 Ala/Ala, as compared to those who carried the GSTO1 140 Ala/Asp and Asp/Asp genotype. Urinary MMA%

levels were signiﬁcantly different within three groups of GSTO2 Asn142Asp genotype (p = 0.01).

The standard concentration of arsenic in drinking water and the risk of low doses of arsenic have been extensively discussed. In 2000, the allowable arsenic level in drinking water was decreased from 50 to 10μg/L in Taiwan, earlier than other most countries. According to the Taipei Water Department of the Taipei City Government, the average arsenic concentration in Taipei tap water is 0.7μg/L (range from non-detectable to 4.0μg/L). However, we randomly collected drinking water from 37 UC cases and measured the total arsenic level; the mean±standard error was 17.14±0.55μg/L. The sum of urinary iAs3±_{, iAs}5±_{, MMA}5±_{and DMA}5±_of

study participants in the present study was lower than those in our previous study (mean value of total arsenic 25μg/ L vs. 70 μg/ L) (Hsueh

et al., 1997). Nonetheless, we still observed a signiﬁcantly increased risk of

UC in those with unfavorable urinary arsenic proﬁle, including higher total arsenic, or higher inorganic arsenic (%), or higher MMA (%) or lower DMA (%).

When mammals are exposed to inorganic arsenate it is reduced to arsenite. Then, arsenite is enzymatically methylated to monomethyl arsenic (MMA) and dimethylarsenic (DMA). Finally, PNP protein or MMA(V) reductase (GSTO1) protein catalyze the conversion of iAs5+_to

MMA5+_{or MMA}5+_{to DMA}5+_{, an arsenic species with an oxidation state}

Table 2

Frequency of the GSTO1 and GSTO2 genotypes and the association between gene polymorphisms of arsenic-metabolism-related enzymes and UC risk.

UC cases (n = 149) no. (%) Control (n = 251) no. (%) Age–gender adjusted OR (95% CI) p Multivariate adjusted OR (95% CI) p GSTO1 Ala140Aspa

Ala/Ala 107(71.81) 166(66.14) 1.00 1.00

Ala/Asp 41(27.52) 78(31.08) 0.82 (0.52–1.29) 0.39 0.70 (0.42–1.18) 0.18 Asp/Asp 1(0.67) 7(2.79) 0.22 (0.03–1.83) 0.16 0.21 (0.02–2.01) 0.18 Ala/Asp + Asp/Asp (vs. Ala/Ala) 42(28.19) 85(33.86) 0.77 (0.49–1.20) 0.25 0.66 (0.40–1.10) 0.11 Ala/Ala + Ala/Asp (vs. Asp/Asp) 148(99.33) 244(97.21) 0.24 (0.03–1.94) 0.18 0.23 (0.02–2.22) 0.20 GSTO1 Glu208Lysb Glu/Glu 145(97.32) 247(98.41) 1.00 1.00 Glu/Lys 4(2.68) 4(1.59) 1.68 (0.41–6.85) 0.47 2.34 (0.49–11.11) 0.28 GSTO2 Asn142Aspc Asn/Asn 88(59.06) 134(53.39) 1.00 1.00 Asn/Asp 59(39.60) 104(41.43) 0.88 (0.58–1.33) 0.53 0.85 (0.53–1.38) 0.52 Asp/Asp 2(1.34) 13(5.18) 0.24 (0.05–1.08) 0.06 0.17 (0.03–0.88) 0.03 Asn/Asp + Asp/Asp (vs. Asn/Asn) 61(40.94) 117(46.61) 0.80 (0.53–1.21) 0.30 0.76 (0.48–1.23) 0.26 Asn/Asn + Asn/Asp (vs. Asp/Asp) 147(98.66) 238(94.82) 0.25 (0.06–1.13) 0.07 0.19 (0.04–0.93) 0.04 Multivariate ORs were adjusted for the highest educational level, paternal ethnicity, cumulative cigarette smoking, alcohol drinking and pesticide usage.

a

GSTO1 140Ala/Ala: the wild type; 140Ala/Asp: the heterozygote genotype; and 140Asp/Asp: homogeneous variant genotype.

b

GSTO1 208Glu/Glu: the wild type; 208Glu/Lys: the heterozygote genotype; and 208Lys/Lys: homogeneous variant genotype.

c

GSTO2 142 Asn/Asn: the wild type; 142 Asn/Asp: the heterozygote genotype; and 142Asp/Asp: homogeneous variant genotype.

Table 3

Median ± standard error of urinary arsenic proﬁles for different genotypes in controls.

n Total arsenic pb _InAs% _pb _MMA% _pb _DMA% _pb GSTO1 Ala140Aspc Ala/Ala 166 15.94 ± 1.14 0.72a 4.32 ± 0.62 0.81a 6.23 ± 0.51 0.06a 88.33 ± 0.84 0.64a Ala/Asp 78 17 ± 1.69 3.79 ± 1.48 4.03 ± 0.98 89.12 ± 1.63 Asp/Asp 7 20.11 ± 4.90 3.48 ± 3.79 0 ± 2.32 84.04 ± 3.58

Ala/Asp + Asp/Asp (vs. Ala/Ala) 85 17.25 ± 1.60 0.51 3.48 ± 1.39 0.53 3.73 ± 0.91 0.02 88.92 ± 1.52 0.48 Ala/Ala + Ala/Asp (vs. Asp/Asp) 244 16.38 ± 0.95 0.71 4.26 ± 0.64 0.55 5.77 ± 0.47 0.30 88.80 ± 0.77 0.65 GSTO1 Glu208Lysd Glu/Glu 247 16.47 ± 0.93 0.54 4.11 ± 0.64 0.07 5.76 ± 0.47 0.83 88.86 ± 0.77 0.54 Glu/Lys 4 22.85 ± 8.71 8.92 ± 1.77 5.00 ± 3.00 88.55 ± 3.77 GSTO2 Asn142Aspe Asn/Asn 134 15.94 ± 1.30 0.75a 4.36 ± 0.71 0.98a 6.46 ± 0.67 0.01a 87.52 ± 1.00 0.29a Asn/Asp 104 16.65 ± 1.41 3.49 ± 1.19 3.76 ± 0.62 89.47 ± 1.24 Asp/Asp 13 17.25 ± 3.90 4.11 ± 2.12 6.52 ± 2.34 84.04 ± 2.72

Asn/Asp + Asp/Asp (vs. Asn/Asn) 117 16.74 ± 1.32 0.46 3.52 ± 1.08 0.96 4.56 ± 0.61 0.30 89.01 ± 1.14 0.38 Asn/Asn + Asn/Asp (vs. Asp/Asp) 238 16.38 ± 0.96 0.99 4.29 ± 0.65 0.87 5.59 ± 0.47 0.62 88.87 ± 0.78 0.30

a_{Calculated by the Kruskal–Wallis test.} b _{Calculated by the Wilcoxon two-sample test.} c

GSTO1 140Ala/Ala: the wild type; 140Ala/Asp: the heterozygote genotype; and 140Asp/Asp: homogeneous variant genotype.

d

GSTO1 208Glu/Glu: the wild type; 208Glu/Lys: the heterozygote genotype; and 208Lys/Lys: homogeneous variant genotype.

e

(5)

of +5 to +3 (Aposhian et al., 2004). The MMA5+_{reducing activity of}

GSTO1 was established from an in vitro study (Mukherjee et al., 2006). For GSTO1 gene knockout mice, the MMA5+_{reducing activity of liver}

cytosol was estimated to be only 20% of that found in wild-type mice

(Mukherjee et al., 2006), which may might point out the important

function of the GSTO1 gene on arsenic metabolism. The major function of GSTO1 and GSTO2 could catalyze the conversion of the arsenic species with an oxidation state of +5 to +3; furthermore, the conversions of trivalent arsenicals to methylated pentavalent arsenic species were through the enzyme of AS3MT (Drobna et al., 2004, 2005, 2006;

Chowdhury et al., 2006; Radabaugh et al., 2002). The trivalent arsenicals,

particularly the trivalent methylated arsenic metabolites, have been identiﬁed as the most toxic forms of arsenic and the toxicological effects of arsenic on urothelium have been established (Eblin et al., 2008; Sens

et al., 2004; Styblo et al., 2000). Although we did not measure the levels

of urinary trivalent arsenicals, we supposed that the levels of urinary pentavalent arsenicals could reﬂect the certain extent of trivalent arsenicals (Del Razo et al., 2001; Francesconi and Kuehnelt, 2004). Therefore, we went on to further clarify whether the polymorphisms of arsenic-related metabolizing enzymes affect individual capacity of arsenic metabolism in human body.

Genes encoding GSTO1/ GSTO2 mapped to chromosomes 10q24.3

(Wood et al., 2006). Recently, many studies have veriﬁed several SNPs

and researchers attempted to explore the connection between these SNPs and urinary arsenic profiles in human studies (Fujihara et al., 2007; Lindberg et al., 2007; Marnell et al., 2003; Paiva et al., 2008; Yu et al., 2003). Until now, there were no candidate gene polymorphisms of GSTO1 or GSTO2 reported and few studies were carried out to elucidate the correlation of these genotypes and urinary arsenic profiles. Paiva et al. measured the urinary arsenic species of 205 Chilean males with occupational arsenic exposure and observed the wide range of total urinary arsenic concentration of 0_{–600 μg/L. Among different gene} polymorphisms of GSTO1 Ala140Asp, Glu155Del and Ala236Val, the GSTO1 Ala236Val heterozygote type was found to be associated with decreased urinary DMA% (p≤0.045) (Paiva et al., 2008). However, 100 Vietnamese people with GSTO1 Glu155del heterozygote type had significantly higher As5+_{level than those with the wild homogenous}

type (Agusa et al., 2010). Conﬂicting results of other studies existed between GSTO1 Ala236Val and urinary arsenic proﬁles (Meza et al.,

2007; Yu et al., 2003; Marnell et al., 2003). Xu et al. recruited 204 subjects

from Inner Mongolia, China who were chronically exposed to arsenic in drinking water and found that the variant allele frequency of GSTO1 Ala140Asp or GSTO2 Asn142Asp was 0.17 and 0.25, respectively. These were close to 0.18 and 0.26 of GSTO1 Ala140Asp or GSTO2 Asn142Asp in our study. However, Xu et al. further analyzed urinary arsenic pro_files but showed no association between the polymorphisms of GSTO1 Ala140Asp or GSTO2 Asn142Asp and urinary arsenic profiles in their results (Xu et al., 2009). In our study, people who carried the wild type of GSTO1 140 Ala/Ala had significantly increased MMA% compared to those carrying the heterozygote or variant homogenous genotype. In addition, urinary MMA% levels were significantly different among the three groups of GSTO2 Asn142Asp genotype (p = 0.01). The urinary MMA% level was decreased in people who carried heterozygous genotype of GSTO2 142 Asn/Asp. However we did not observe the lower urinary MMA% in people who carried the homogenous variant genotype of GSTO2 142 Asp/Asp compared to those who carried the wild type of GSTO2 142 Asn/Asn. Additional subjects are needed for further investigation. In addition, De Chaudhuri et al. recruited 229 patients of skin cancer and 199 controls exposed to similar levels of arsenic in their drinking water and to elucidate the association between the polymor-phisms of AS3MT, PNP, GSTO1 and GSTO2 and arsenic-associated skin lesions (De Chaudhuri et al., 2008). In their study, the polymorphisms of PNP His20Hsi, Gly51Ser and Pro57Pro were significantly associated with arsenic-associated skin lesions, but no association was found between gene polymorphisms of GSTO1 and GSTO2 and arsenic-associated skin lesions. Although the important roles of PNP other than GSTOs on

urinary arsenic proﬁles have been addressed (De Chaudhuri et al., 2008), the functional role of PNP for reduction of arsenate to arsenite is still not clear (Nemeti et al., 2003; Radabaugh et al, 2002). In our study, we showed the homogenous variant genotype of GSTO2 142 Asp/Asp was inversely associated with UC risk. However we did not directly observe the association between the polymorphisms of GSTO2 Asn142Asp and UC risk through the change of urinary arsenic proﬁle.

After exclusion of those of incomplete biological specimen collection or questionnaire information, there was a relatively small sample size in our study; however, there were no differences in other covariates, such as genotyping or urinary total arsenic levels (data not shown) between people included and excluded from analysis. While the enzyme activity of GSTO1 and GSTO2 was unknown in our studies; we still observed differences in urinary MMA% among varied genotypes of GSTO1 and GSTO2. Furthermore, ourﬁndings showed a signiﬁcant protective effect of the polymorphism of GSTO2 Asn142Asp on UC risk. Large-scale studies may be required to verify the association between the single nucleotide polymorphisms of arsenic-metabolism-related enzymes and UC risk. Acknowledgments

The study was supported by grants from the National Science Council of the ROC (NSC 86-2314-B-038-038, NSC 87-2314-B-038-029, NSC 88-2314-B-038-112, NSC 89-2314-B038-049, SC-89-2320-B038-013, NSC 90-2320-B-038-021, NSC 91-3112-B-038-0019, NSC 92-3112-B-038-001, NSC 93-3112-B-038-001, NSC 94-2314-B-038-023, NSC 95-2314-B-038-007, NSC 96-2314-B038-003, NSC 97-2314-B-038-015-MY3 (1-3), and NSC 97-2314-B-038-015-MY3 (2-3)). References

Agusa T, Iwata H, Fujihara J, Kunito T, Takeshita H, Minh TB, et al. Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam. Toxicol Appl Pharmacol 2010;242:352–62.

Aposhian HV, Aposhian MM. Arsenic toxicology: Five questions. Chem Res Toxicol 2006;19:1-15.

Aposhian HV, Zakharyan RA, Avram MD, Sampayo-Reyes A, Wollenberg ML. A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species. Toxicol Appl Pharmacol 2004;198:327–35.

Chen CJ, Kuo TL, Wu MM. Arsenic and cancers. Lancet 1988;1:414–5.

Chen YC, Amarasiriwardena CJ, Hsueh YM, Christiani DC. Stability of arsenic species and insoluble arsenic in human urine. Cancer Epidemiol Biomarkers Prev 2002;11:1427–33. Chen YC, Su HJ, Guo YL, Hsueh YM, Smith TJ, Ryan LM, et al. Arsenic methylation and

bladder cancer risk in Taiwan. Cancer Causes Control 2003;14:303–10. Chiou HY, Hsueh YM, Liaw KF, Horng SF, Chiang MH, Pu YS, et al. Incidence of internal

cancers and ingested inorganic arsenic: A seven-year follow-up study in Taiwan. Cancer Res 1995;55:1296–300.

Chowdhury UK, Zakharyan RA, Hernandez A, Avram MD, Kopplin MJ, Aposhian HV. Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration. Toxicol Appl Pharmacol 2006;216:446–57.

Cohen SM, Arnold LL, Eldan M, Lewis AS, Beck BD. Methylated arsenicals: The implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit Rev Toxicol 2006;36:99-133.

De Chaudhuri S, Ghosh P, Sarma N, Majumdar P, Sau TJ, Basu S, et al. Genetic variants associated with arsenic susceptibility: Study of purine nucleoside phosphorylase, arsenic (+3) methyltransferase, and glutathione s-transferase omega genes. Environ Health Perspect 2008;116:501–5.

Del Razo LM, Styblo M, Cullen WR, Thomas DJ. Determination of trivalent methylated arsenicals in biological matrices. Toxicol Appl Pharmacol 2001;174:282–93. Drobna Z, Waters SB, Devesa V, Harmon AW, Thomas DJ, Styblo M. Metabolism and

toxicity of arsenic in human urothelial cells expressing rat arsenic (+3 oxidation state)-methyltransferase. Toxicol Appl Pharmacol 2005;207:147–59.

Drobna Z, Waters SB, Walton FS, LeCluyse EL, Thomas DJ, Styblo M. Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes. Toxicol Appl Pharmacol 2004;201:166–77.

Drobna Z, Xing W, Thomas DJ, Styblo M. shRNA silencing of AS3MT expression minimizes arsenic methylation capacity of HepG2 cells. Chem Res Toxicol 2006;19:894–8. Eblin KE, Bredfeldt TG, Gandolﬁ AJ. Immortalized human urothelial cells as a model of

arsenic-induced bladder cancer. Toxicology 2008;248:67–76.

Francesconi KA, Kuehnelt D. Determination of arsenic species: A critical review of methods and applications, 2000-2003. Analyst 2004;129:373–95.

Fujihara J, Kunito T, Takeshita H. Frequency of two human glutathione-S-transferase omega-1 polymorphisms (E155 deletion and E208K) in Ovambo and Japanese populations using the PCR-based genotyping method. Clin Chem Lab Med 2007;45:621–4.

(6)

Hsueh YM, Chiou HY, Huang YL, Wu WL, Huang CC, Yang MH, et al. Serum beta-carotene level, arsenic methylation capability, and incidence of skin cancer. Cancer Epidemiol Biomarkers Prev 1997;6:589–96.

Hsueh YM, Huang YL, Huang CC, Wu WL, Chen HM, Yang MH, et al. Urinary levels of inorganic and organic arsenic metabolites among residents in an arseniasis-hyperendemic area in Taiwan. J Toxicol Environ Health 1998;54:431–44.

Hsueh YM, Hsu MK, Chiou HY, Yang MH, Huang CC, Chen CJ. Urinary arsenic speciation in subjects with or without restriction from seafood dietary intake. Toxicol Lett 2002;133: 83–91.

Kolsch H, Larionov S, Dedeck O, Orantes M, Birkenmeier G, Grifﬁn WS, et al. Association of the glutathione S-transferase omega-1 Ala140Asp polymorphism with cerebrovascular atherosclerosis and plaque-associated interleukin-1 alpha expression. Stroke 2007;38: 2847–50.

Leite JL, Morari EC, Granja F, Campos GM, Guilhen AC, Ward LS. Inﬂuence of the glutathione s-transferase gene polymorphisms on the susceptibility to basal cell skin carcinoma. Rev Med Chil 2007;135:301–6.

Lin SM. Diagnostic usefulness of trace arsenic in human urine, whole blood, hair and ﬁngernails. Gaoxiong Yi Xue Ke Xue Za Zhi 1986;2:100–113.

Lin S, Shi Q, Nix FB, Styblo M, Beck MA, Herbin-Davis KM, et al. A novel S-adenosyl-L-methionine:arsenic(III) methyltransferase from rat liver cytosol. J Biol Chem 2002;277: 10795–803.

Lindberg AL, Kumar R, Goessler W, Thirumaran R, Gurzau E, Koppova K, et al. Metabolism of low-dose inorganic arsenic in a central European population: Inﬂuence of sex and genetic polymorphisms. Environ Health Perspect 2007;115:1081–6.

Ma M, Le XC. Effect of arsenosugar ingestion on urinary arsenic speciation. Clin Chem 1998;44:539–50.

Marahatta SB, Punyarit P, Bhudisawasdi V, Paupairoj A, Wongkham S, Petmitr S. Polymorphism of glutathione S-transferase omega gene and risk of cancer. Cancer Lett 2006;236:276–81.

Marnell LL, Garcia-Vargas GG, Chowdhury UK, Zakharyan RA, Walsh B, Avram MD, et al. Polymorphisms in the human monomethylarsonic acid (MMA V) reductase/hGSTO1 gene and changes in urinary arsenic proﬁles. Chem Res Toxicol 2003;16:1507–13. Meza M, Gandolﬁ AJ, Klimecki WT. Developmental and genetic modulation of arsenic

biotransformation: A gene by environment interaction? Toxicol Appl Pharmacol 2007;222:381–7.

Mukherjee B, Salavaggione OE, Pelleymounter LL, Moon I, Eckloff BW, Schaid DJ, et al. Glutathione S-transferase omega 1 and omega 2 pharmacogenomics. Drug Metab Dispos 2006;34:1237–46.

Negri E, La VC. Epidemiology and prevention of bladder cancer. Eur J Cancer Prev 2001;10:7-14.

Nemeti B, Csanaky I, Gregus Z. Arsenate reduction in human erythrocytes and rats: Testing the role of purine nucleoside phosphorylase. Toxicol Sci 2003;74:22–31. Paiva L, Marcos R, Creus A, Coggan M, Oakley AJ, Board PG. Polymorphism of glutathione

transferase Omega 1 in a population exposed to a high environmental arsenic burden. Pharmacogenet Genomics 2008;18:1-10.

Pu YS, Yang SM, Huang YK, Chung CJ, Huang SK, Chiu AW, et al. Urinary arsenic proﬁle affects the risk of urothelial carcinoma even at low arsenic exposure. Toxicol Appl Pharmacol 2007;218:99-106.

Radabaugh TR, Sampayo-Reyes A, Zakharyan RA, Aposhian HV. Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem Res Toxicol 2002;15:692–8.

Schmuck EM, Board PG, Whitbread AK, Tetlow N, Cavanaugh JA, Blackburn AC, et al. Characterization of the monomethylarsonate reductase and dehydroascorbate reductase activities of Omega class glutathione transferase variants: Implications for arsenic metabolism and the age-at-onset of Alzheimer's and Parkinson's diseases. Pharmacogenet Genomics 2005;15:493–501.

Sens DA, Park S, Gurel V, Sens MA, Garrett SH, Somji S. Inorganic cadmium- and arsenite-induced malignant transformation of human bladder urothelial cells. Toxicol Sci 2004;79:56–63.

Simeonova PP, Wang S, Toriuma W, Kommineni V, Matheson J, Unimye N, et al. Arsenic mediates cell proliferation and gene expression in the bladder epithelium: Association with activating protein-1 transactivation. Cancer Res 2000;60:3445–53.

Steinmaus C, Bates MN, Yuan Y, Kalman D, Atallah R, Rey OA, et al. Arsenic methylation and bladder cancer risk in case-control studies in Argentina and the United States. J Occup Environ Med 2006;48:478–88.

Styblo M, Del Razo LM, Vega L, Germolec DR, LeCluyse EL, Hamilton GA, et al. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch Toxicol 2000;74:289–99.

Su PF, Hu YJ, Ho IC, Cheng YM, Lee TC. Distinct gene expression proﬁles in immortalized human urothelial cells exposed to inorganic arsenite and its methylated trivalent metabolites. Environ Health Perspect 2006;114:394–403.

Thomas DJ, Li J, Waters SB, Xing W, Adair BM, Drobna Z, et al. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals. Exp Biol Med (Maywood) 2007;232:3-13.

Wahner AD, Glatt CE, Bronstein JM, Ritz B. Glutathione S-transferase mu, omega, pi, and theta class variants and smoking in Parkinson's disease. Neurosci Lett 2007;413:274–8. Wood TC, Salavagionne OE, Mukherjee B, Wang L, Klumpp AF, Thomae BA, et al. Human

arsenic methyltransferase (AS3MT) pharmacogenetics: Gene resequencing and functional genomics studies. J Biol Chem 2006;281:7364–73.

Xu Y, Li X, Zheng Q, Wang H, Wang Y, Sun G. Lack of association of glutathione-S-transferase omega 1(A140D) and omega 2 (N142D) gene polymorphisms with urinary arsenic proﬁle and oxidative stress status in arsenic-exposed population. Mutat Res 2009;679: 44–9.

Yu L, Kalla K, Guthrie E, Vidrine A, Klimecki WT. Genetic variation in genes associated with arsenic metabolism: Glutathione S-transferase omega 1-1 and purine nucleoside phosphorylase polymorphisms in European and indigenous Americans. Environ Health Perspect 2003;111:1421–7.