Environmental Health Perspectives • volume 118 | number 11 | November 2010 

### 1545 

# Research

Chlorine is a cost-effective drinking water dis

infectant that has been used since the early twentieth century to control a panoply of

water borne infectious diseases. By

- products of the inter action of chlorine with organic pre

cursors in water were first noted in 1974, with the discovery of trihalomethanes (THMs) in disinfected water (Bellar and Lichtenberg 1974; Rook 1974). Since then, hundreds of halogenated chemical species in the disinfec

tion by

- product (DBP) mixture have been detected, including both brominated and chlorinated compounds (Richardson 2003). THMs and haloacetic acids (HAAs) are the chemical groups at highest concentration in most by

- product mixtures. Toxicological and epidemiologic studies of DBPs provide evi

dence of elevated risk of cancer and adverse birth outcomes (Cantor et al. 2006; Grellier

et al. 2010; Nieuwenhuijsen et al. 2009). In particular, human bladder cancer has been consistently linked with long-term exposure (Cantor et al. 1998; King and Marrett 1996; McGeehin et al. 1993; Villanueva et al. 2004). These observations are supported by evidence of mutagenicity of the mixture and carcino

genicity of some constituents (Komulainen 2004; Richardson et al. 2007). To date, the role of genetic variability in modulating adverse health effects of DBPs has received limited attention (Infante-Rivard et al. 2002), and bladder cancer has not been studied in this regard. At least three enzymes in the metabolic pathways of DBP components are candidates for examination. Glutathione S

- transferase (GST) theta

- 1 (GSTT1) activates bromi

nated THMs to mutagens in a transgenic strain of Salmonella ( DeMarini et al. 1997; Pegram et al. 1997). GST zeta

- 1 (GSTZ1) catalyzes the oxygenation of dichloro- and other α -haloacids, some of which are animal
carcino gens (DeAngelo et al. 1999; Melnick

et al. 2007; Tong et al. 1998). Cytochrome P450 2E1 ( CYP2E1 ) metabolizes a wide vari

ety of aliphatic hydro carbons, solvents, and industrial monomers (Guengerich et al. 1991) and is responsible for the primary oxidation of THMs. Genes that code for these enzymes are polymorphic in human populations, with the GSTT1 deletion resulting in lack of enzyme activity

, and with several non synonymous single-nucleotide polymorphisms (SNPs) in GSTZ1 and CYP2E1 resulting in modified enzymatic activity (Blackburn et al. 2001; Bolt et al. 2003). We hypothesized that one or more of these functional polymorphisms could influence bladder cancer risk posed by DBPs, and we investigated this in a large case

– control study in Spain, where in a previous study (Villanueva et al. 2007) we observed elevated risk of bladder cancer after long-term exposure to DBPs.


### Materials and Methods We conducted a hospital-based case

– control study in 18 hospitals located in five areas of Spain [ Asturias, Barcelona metropolitan area, Valles/Bages (including the municipali

ties of Manresa and Sabadell), Alicante, and Tenerife ] (Appendix 1) . Eligible cases were
21–80 years of age, newly diagnosed with histologically confirmed urothelial carcinoma of the bladder between 1998 and 2001, and living in the catchment geographic area of

Address correspondence to K.P. Cantor, National Cancer Institute, 8109 EPS, 6120 Executive Blvd., Bethesda, MD 20892-7240 USA. Telephone: (301) 435-4718. Fax: (301) 402-1819. E-mail: cantork@ nih.gov

Supplemental Material is available online (doi:10. 1289/ehp.1002206 via http://dx.doi.org/).

We thank F. Fernández, Centre for Research in Environmental Epidemiology; R.C. Saal, Westat, Inc.; and L. Carroll and E. Boyd, Information Management Services Inc., for technical assistance.

This work was funded by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (N02-CP-11015), the Fondo de Investigación Sanitaria (00/0745, G03/174, G03/160, C03/09, and C03/90), and the Instituto de Salud Carlos III, Spanish Health Ministry (CP06/00341).

The authors declare they have no actual or potential competing financial interests.

Received 24 March 2010; accepted 30 July 2010. 

## Polymorphisms in GSTT1

, GSTZ1 , and CYP2E1 , Disinfection By-products, and Risk of Bladder Cancer in Spain

Kenneth P. Cantor, 1,2 Cristina M. Villanueva, 3,4,5 Debra T. Silverman, 1 Jonine D. Figueroa, 1 Francisco X. Real, 4,6,7 Monserrat Garcia-Closas, 1 Nuria Malats, 4,6 Stephen Chanock, 1 Meredith Yeager, 1 Adonina Tardon, 8 Reina Garcia-Closas, 9 Consol Serra, 7,10 Alfredo Carrato, 11 Gemma Castaño-Vinyals, 3,4,5 Claudine Samanic, 1 Nathaniel Rothman, 1 and Manolis Kogevinas 3,4,5,12

1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA; 2 KP Cantor Environmental LLC, Silver Spring, Maryland, USA; 3 Centre for Research in Environmental Epidemiology, Barcelona, Spain; 4 Institut Municipal d’Investigacio Medica–Hospital del Mar, Barcelona, Spain; 5 CIBER Epidemiologia y Salud Publica, Barcelona, Spain; 6 Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; 7 Universidad Pompeu Fabra, Barcelona, Spain; 8 Universidad de Oviedo, Oviedo, Spain;
9 Unidad de Invesigación, Hospital Universitario de Canarias, La Laguna, Spain; 10 Consorci Hospitalari Parc Tauli, Sabadell, Spain;
11 Ramon y Cajal University Hospital, Madrid, Spain; 12 National School of Public Health, Athens, Greece

B ackground : Bladder cancer has been linked with long-term exposure to disinfection by- products (DBPs) in drinking water.

O b jectives : In this study we investigated the combined influence of DBP exposure and poly

morphisms in glutathione S -transferase ( GSTT1
, GSTZ1 ) and cytochrome P450 ( CYP2E1 ) genes in the metabolic pathways of selected by

- products on bladder cancer in a hospital-based case

– control study in Spain.

M ethods : Average exposures to trihalomethanes (THMs ; a surrogate for DBPs) from 15 years of

age were estimated for each subject based on residential history and information on municipal water sources among 680 cases and 714 controls. We estimated effects of THMs and GSTT1
, GSTZ1
, and CYP2E1 polymorphisms on bladder cancer using adjusted logistic regression models with and without interaction terms.

R

esults : THM exposure was positively associated with bladder cancer: adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were 1.2 (0.8–1.8), 1.8 (1.1–2.9), and 1.8 (0.9–3.5) for THM quartiles 2, 3, and 4, respectively, relative to quartile 1. Associations between THMs and bladder cancer were stronger among subjects who were GSTT1 +/+ or +/– versus GSTT1 null ( p interaction

= 0.021), GSTZ1 rs1046428 CT/TT versus CC ( p interaction = 0.018), or CYP2E1 rs2031920 CC versus CT/TT ( p interaction = 0.035). Among the 195 cases and 192 controls with high- risk forms of GSTT1 and GSTZ1, the ORs for quartiles 2, 3, and 4 of THMs were 1.5 (0.7–3.5), 3.4 (1.4–8.2), and 5.9 (1.8–19.0), respectively.

C onclusions : Polymorphisms in key metabolizing enzymes modified DBP-associated bladder can

cer risk. The consistency of these findings with experimental observations of GSTT1, GSTZ1, and CYP2E1 activity strengthens the hypothesis that DBPs cause bladder cancer and suggests possible mechanisms as well as the classes of compounds likely to be implicated.

K

ey words : bladder cancer, CYP2E1 , disinfection by

- products, drinking water, GSTT1
, GSTZ1
, trihalomethanes. Environ Health Perspect 118:1545–1550 (2010). doi:10.1289/ehp.1002206 [Online 12 September 2010]Cantor et al.



### 1546 volume 118 | number 11 | November 2010 • Environmental Health Perspectives participating hospitals. Cases were identified from the registers of urological services aug

mented by regular and frequent evaluations of hospital discharge records, pathology records

, and local cancer registries. A panel of expert pathologists confirmed diagnoses and ensured uniformity of classification criteria, based on the 1998 World Health Organization/ International Society of Urological Pathology system (Epstein et al. 1998). Controls were selected from patients admitted to participating hospitals with con

ditions thought to be unrelated to the major risk factors of bladder cancer, such as tobacco use. Diagnostic categories for controls were as follows: 37% hernias, 11% other abdominal surgery, 23% fractures, 7% other orthopedic problems, 12% hydrocoele, 4% circulatory disorders, 2% dermatological disorders, 1% ophthal mologic al disorders, and 3% other diseases. Controls were individually matched to cases by age at interview within 5

- year strata, sex, ethnic origin, and hospital catch

ment area, a well- defined area corresponding to the specific health services region covered by each hospital. Written informed consent was obtained from each subject before the study. The study was approved by the review board of each participating institution and in accord with an assurance filed with and approved by the U.S. Department of Health and Human Services. Individual data. After obtaining informed consent, trained interviewers administered a computer-assisted personal interview (CAPI) to participants during their hospital stay. Interview items included socio demographic characteristics; smoking habits; occupational, residential, and medical histories; and familial history of cancer. We identified 1,457 eligible cases and 1,465 eligible controls. Of these, 84% of cases ( n = 1,219) and 87% of con

trols ( n = 1,271) participated. Subjects who refused to answer the CAPI were administered a reduced interview of critical items (21% of cases and 19% of controls). Questionnaire information on water-related exposures used in this analysis included residential history from birth [all residences of at least 1 year, drinking water source at each residence (municipal/bot

tled/private well/other)] and swimming pool use as an adult. These data were collected from all participants, including those who responded to the critical items questionnaire. Exposure data. Using a structured ques

tionnaire, we collected historic water quality data from approximately 200 local authori

ties and 150 water companies in the study areas. For 123 study municipalities, covering 78.5% of the total study exposure-years, we obtained annual average THM levels. In addi

tion, one of us (C .M. V

. ) measured levels of the four THMs [chloroform, bromo dichloro

methane (BDCM), dibromo chloro methane

, and bromo form] in 113 tap water samples from the studied geographic areas between September and December 1999. Average THM levels in recent years were extrapolated back to approximately 1920. Historical THM levels were estimated by municipality under the assumption that past THM levels were similar to current concentra

tions when the water source had not changed. When the water source had changed, we cal

culated the average THM level using the pro

portion of surface water during the rele vant time period. We assumed that the THM level before the start of chlorination was zero. The exposure assessment was described previously (Villanueva et al. 2006, 2007). Lifetime individual exposure indices. We merged individual and municipal databases with individual residential information by yearand municipality, and obtained individual yearby-year average THM levels, water source, and chlorination status for each study subject. We used as an exposure metric the average THM level of the water source serving participant residences in the period between age 15 years and the interview, as described previously in the analysis of the main effects of DBP expo

sure (Villanueva et al. 2007). As in that study, we restricted our analysis to subjects with a household THM estimate for at least 70% of the years in this exposure window. Genotyping. DNA for genotype assays was extracted from leukocytes or mouthwashsamples as described previously (GarciaClosas et al. 2005). Genotype assays for polymorphisms in GSTT1

, GSTM1 , and N

- acetyltransferase 2 ( NAT2 ) were con

ducted at the Core Genotyping Facility of the Division of Cancer Epidemiology and Genetics (National Cancer Institute ) . The SNPs of NAT2 and assignments of rapid/intermediate/ slow acetylator types, as well as the GSTM1 genotypes, have been described in detail else

where (Garcia-Closas et al. 2005). GSTT1 genotypes were defined as null (

– /–) if a dele

tion was found in both copies of the gene and present if one (+/–) or none (+/+) of the copies had a deletion. We used the TaqMan assay (Applied Biosystems, Foster City, CA, USA) for SNPs in NAT2 , two of three SNPs in GSTZ1 [ E32K (rs7975) and G42R (rs7972) ], and deletions in GSTT1 and GSTM1 . The methods for each specific assay are available from the National Cancer Institute ( 2010). A third SNP in GSTZ1 (T82M rs1046428) and three SNPs in CYP2E1 (IVS7–118C>G rs2070676

, –1054C>T rs2031920

, and –1514G>T rs8192766) were determined in a GoldenGate assay (Illumina, San Diego, CA

, USA ) (Garcia-Closas et al. 2007). All geno

types studied were in Hardy-Weinberg equi

librium in the control population. Duplicate quality control samples ( n = 93 pairs) showed

≥ 99% agreement for all assays. Statistical analysis. We considered results to be statistically significant if the alpha level

was ≤ 0.05. Subjects were grouped by quartile of the time-weighted average THM level since age 15 years. We used unconditional logistic regression to calculate odds ratios (OR s ) and 95% confidence intervals (CI s ). OR s were adjusted for age (continuous), sex, smoking sta

tus (never/former/current)

, size of the munici

pality of longest residence until 18 years of age (reported by the participant as metropolis/city, small city, village, or farm), education (three strata: less than primary school, less than high school, and high school or more), geographic area (six strata: Barcelona, Sabadell, Manresa, Alicante, Tenerife, and Asturias)

, and overall quality of interview (reported by interviewer as unsatisfactory, questionable, reliable

, or high quality). Analyses using more detailed smoking information (total duration, aver

age daily number of cigarettes, tobacco type, pack-years) gave similar results and are not reported. Subjects were defined as “ former ” smokers if they had quit at least 1 year before the interview. Missing data for covariates were coded to a separate cate gory for each variable included in the models. This applied to a small proportion of respondents for each covariate. For example, < 1% of respondents were miss

ing data on smoking status (Samanic et al. 2006 ). Linear p -trends were calculated using a likelihood ratio test comparing the model with and without the THM exposure variable mod

eled as a continuous variable with each quartile coded according to its median value. Interactions between genotypes and expo

sure to DBPs , as estimated by average THM levels serving the household between 15 years of age and diagnosis (cases) or interview (con

trols), were also tested using the likelihood-ra

tio test to allow estimation of parameters under the assumption of genotype

– DBP (THMs) independence in the source population. Tests for multiplicative inter action were used to assess whether the genotype OR s within categories of DBP exposure or

, equivalently, DBP OR s within geno type categories differed significantly from each other. Haplotype fre

quencies for GSTZ1 and CYP2E1 were esti

mated using HaploStats (version 1.2.1; http:// mayoresearch. mayo.edu/mayo/research/ biostat/ schaid.cfm) using the program language R (http://www.r-project.org/).



### Results
Of the 1,219 cases and 1,271 controls inter

viewed, 1,188 (97%) cases and 1,173 (92%) controls provided a blood or buccal cell sample for DNA extraction. After excluding cases and controls with low amounts of DNA, non white individuals (to limit hetero geneity), and DNA quality control difficulties, the study popu

lation with adequate genetic material avail

able for analysis numbered 1,150 cases andBladder cancer, DBPs, and genetic polymorphisms

Environmental Health Perspectives • volume 118 | number 11 | November 2010 

### 1547 1,149 controls (Garcia-Closas et al. 2005). After further excluding individuals with inad

equate exposure data as described above, the maximum number s with genotyping data

were 680 cases (595 males, 85 females) and 714 controls (622 males, 92 females). In each gene-specific analysis, we applied additional exclusions because of incomplete genetic information. The ORs (95% CIs) for former [ 2.7 (1.9–3.7) ] and current [ 5.9 (4.2–8.5) ] smok

ers of cigarettes in the analysis data set, rela

tive to never-smokers, paralleled those found in the full study population (Samanic et al. 2006). The ORs (95% CIs) by increasing quartile of THMs were 1.0 (referent), 1.2 (0.8–1.9), 1.8 (1.1–2.9), and 1.8 (0.9–3.5) ( p trend = 0.029) (Table 1), similar to the find

ings of Villanueva et al. (2007). These ORs are adjusted for the co variates listed above, as are all subsequent ORs reported here. Findings from Garcia-Closas et al. (2005), from this study population, showed significant associa

tions between bladder cancer and NAT2 slow acetylator compared with rapid/intermediate
geno types [ OR (95% CI) of 1.4 (1.2–1.7) ] and GSTM1 null ( –/– ) versus GSTM1 pres

ent genotypes [1.7 (1.4–2.0) ]. Estimates in the present analyses were similar, with NAT2 (slow compared with rapid plus inter mediate acetylator type) having an OR (95% CI) of 1.33 (1.06–1.68)

, and with GSTM1 null compared with the GSTM1 present geno type having an OR (95% CI) of 1.77 (1.41–2.22). We found slightly elevated, non significant risks for GSTT1 presence compared with null [1.21 (0.92–1.59) ] . The association for the inverse relationship (null vs

. presence) was 0.81 (0.61–1.09). In the full study, compared with the homozygous presence of GSTT1 , the ORs (95% CIs) were 1.2 (1.0–1.5) for hetero zygotes (+/–) and 1.0 (0.8–1.3) for homo zygous null (

– /–) (Garcia-Closas et al. 2005). We found non significant elevations or decreases in risk for the three functional SNPs of GSTZ1 and the three variants of CYP2E1 that we tested. Associations for the three SNPs
in GSTZ1 and the three in CYP2E1 have not been reported previously. The GSTZ1 SNPs were in linkage dis equilibrium (pairwise D  ́ values ranged from 0.98 to 1.0); however

, they were not highly correlated with each other ( r 2 ranged from 0.02 to 0.18). Of the three CYP2E1 SNPs tested, rs8192766 was not in linkage disequilibrium with rs2070676 ( D  ́
= 0.34). The other two pairs were in linkage dis equilibrium ( D  ́ ≥ 0.98). None of the three CYP2E1 SNPs was highly correlated with any other ( r 2 ranged from 0.006 to 0.34). Haplotype analysis for GSTZ1 and the three SNPs genotyped reveal ed no common haplo

type significantly associated with risk (global
p = 0.80 ; data not shown). We made similar observations for CYP2E1 (global p = 0.76). We observed significant interactions with THMs for GSTT1 (present vs

. null) ( p interaction

= 0.021), exon 7 +29T>C rs1046428 in GSTZ1 ( p interaction = 0.018), and –1054C>T rs2031920 in CYP2E1 ( p interaction = 0.035 ; Table 2) but not for the other two SNPs in GSTZ1 that we tested (rs7975, rs7972) or for the two other SNPs tested in CYP2E1 (rs2070676, rs8192766 ; data not shown). We found no significant trends in associations with increasing THM quartiles among subjects with the GSTT1 null, GSTZ1 CC

, or CYP2E1 CT/TT genotypes; however, we did find sig

nificant positive trends with increasing THM exposure among subjects with the GSTT1
present genotype GSTZ1 CT/TT, or CYP2E1 CC (Table 2). Haplotype analysis for GSTZ1 and CYP2E1 did not provide additional infor

mation beyond the single SNP analysis (data not shown). We found no evidence of inter

action between either NAT2 slow acetylator

or GSTM1 -null genotype and long- term aver

age THM level ( p interaction = 0.95 and 0.79, respectively ; data not shown). Because of small numbers of subjects (37 cases, 42 controls) with the CT variant of CYP2E1 rs2031920 (no subjects carried the TT variant), it was not possible to analyze a three-way interaction that included these subjects. We found a significant inter action between average THM level and combined GSTT1 and GSTZ1 rs1046428 genotypes ( p interaction = 0.0052) in analyses that included **Table 1.** OR s (95% CI s ) for bladder cancer for quartile levels of average long-term level of household THMs and for selected SNPs.

Measure n (cases/ controls) a OR (95% CI) b p -Value Average THM level at home (μg/L, age 15 to index age)

≤ 8.0 156/175 1.0

> 8.0–26.0 153/174 1.2 (0.8–1.9)

> 26.0–49.0 197/169 1.8 (1.1–2.9)

> 49.0 174/196 1.8 (0.9–3.5)
p trend 0.029 Polymorphism
NAT2 Fast 243/299 1.00 Slow 428/403 1.33 (1.06–1.68) 0.01 GSTM1

Present 257/366 1.00 Null 420/346 1.77 (1.41–2.22) < 0.001 GSTT1

Null 136/160 1.00 Present 542/550 1.21 (0.92–1.59) 0.13 GSTZ1

rs1046428, T82M CC 405/402 1.00 CT 213/213 1.06 (0.82–1.35) 0.67 TT 31/29 1.07 (0.61–1.86) 0.82 rs7975, E32K GG 314/330 1.00 AG 275/276 1.06 (0.83–1.34) 0.66 AA 68/63 1.22 (0.82–1.81) 0.33 rs7972, G42R GG 553/572 1.00 AG 100/91 1.16 (0.84–1.61) 0.36 AA 4/6 0.69 (0.18–2.56) 0.57 CYP2E1

rs2070676 CC 479/474 1.00 CG 161/166 0.93 (0.71–1.21) 0.59 GG 9/5 2.03 (0.62–6.62) 0.24 rs2031920 CC 590/569 1.00 CT 37/42 0.76 (0.47–1.23) 0.27 TT 0/0

— rs8192766 GG 528/533 1.0 GT 118/106 1.05 (0.77–1.42) 0.77 TT 3/7 0.32 (0.08–1.30) 0.11

a Numbers of cases or controls per measure may not equal the total number of cases (680) and controls (714) because of missing genotype data. b OR (95% CI) from logistic regression adjusted for age (continuous), sex, smoking status (never/former/current), size of the munic

ipality of longest residence until 18 years of age, edu

cation (three strata), geographic area ( six strata), and overall quality of interview. **Table 2.** Interaction between average THM exposure and polymorphic forms of three genes: GSTT1

, GSTZ1 , and CYP2E1

. a Gene, average THM (μg/L) n (cases/controls) OR (95% CI) n (cases/controls) OR (95% CI) p interaction b

GSTT1 Null Present

≤ 8.0 34/34 1.0 (reference) 121/141 1.0 (reference)

> 8.0–26.0 36/37 1.2 (0.5–2.5) 116/136 1.2 (0.7–1.9)

> 26.0–49.0 37/41 1.2 (0.5–2.5) 160/126 2.0 (1.2–3.4)

> 49.0 29/48 1.0 (0.4–2.5) 145/147 2.2 (1.1–4.3) 0.021
p trend 0.28 0.0072 GSTZ1 rs1046428 CC CT/TT

≤ 8.0 95/86 1.0 (reference) 52/62 1.0 (reference)

> 8.0–26.0 100/102 1.1 (0.7–1.9) 47/54 1.4 (0.7–2.7)

> 26.0–49.0 116/97 1.5 (0.9–2.7) 73/62 2.2 (1.1–4.2)

> 49.0 94/117 1.3 (0.6–2.8) 72/64 2.9 (1.3–6.7) 0.018
p trend 0.28 0.0043 CYP2E1 rs2031920 CT/TT CC

≤ 8.0 15/9 1.0 (reference) 125/132 1.0 (reference)

> 8.0–26.0 10/14 0.98 (0.4–2.5) 133/141 1.3 (0.8–2.0)

> 26.0–49.0 9/11 1.1 (0.4–3.1) 176/134 2.1 (1.2–3.5)

> 49.0 3/8 0.6 (0.1–2.7) 156/162 2.0 (1.0–4.1) 0.035
p trend 0.33 0.014

a OR s (95% CI s ) from logistic regression adjusted for age (continuous), sex, smoking status (never/former/current), size of the municipality of longest residence until 18 years of age , education (three strata), geographic area ( six strata), and overall quality of interview. b p -Value for multiplicative interaction between THM level and the respective polymorphism.Cantor et al.



### 1548 volume 118 | number 11 | November 2010 • Environmental Health Perspectives subjects with both the variant and common forms of CYP2E1 rs2031920 (Table 3). Among subjects with both GSTT1 present and GSTZ1 CT/TT genotypes, OR increased monotonically to 5.9 (95% CI, 1.8–19.0) in the highest quartile of THMs ( p trend = 0.0012 )

. In contrast, among subjects with both GSTT1 null and GSTZ1 CC genotypes, we found no increase in relative risk with increas

ing THM level ( p interaction = 0.0052). These analyses excluded subjects with both low- risk variants of GSTZ1 and high- risk variants of GSTT1 , or vice versa. When we restricted this analysis to subjects with the common form (CC) of CYP2E1 rs2031920, among those with both GSTT1 present and GSTZ1 CT/ TT genotypes, the OR increased monotoni

cally to 9.3 (2.5–34.0) in the highest quartile of THM exposure (> 49.0 μ g/L), relative to participants with these genotypes in the low

est quartile [≤ 8.0 μ g/L ; p trend = 0.0006 with
p interaction = 0.010 ; see Supplemental Material, Table 1 (doi:10. 1289/ehp.1002206)]. We calculated the main effects for the respective SNPs within increasing quartile strata of long-term average THMs (Table 4) where we found increasing relative risks for bladder cancer for GSTT1 present, GSTZ1 rs1046428 CT/TT , and CYP2E1 rs2031920 CC. Among subjects with exposure to THMs in the highest quartile, the ORs were elevated and the 95% CIs excluded 1.0 for GSTT1 present (vs

. GSTT1 null), GSTZ1 (CT/TT vs

. CC), and CYP2E1 (CC vs

. CT/TT), whereas ORs for these polymorphisms were close to 1.0 among subjects in the lowest THM exposure quartile. The associations with bladder cancer risk for NAT2 slow versus rapid/intermediate acetylator and GSTM1 null versus present within each THM stratum were variable and consistent with the overall elevated main effects for these genes. In earlier analyses of these data, we found a significant association with ever swimming in pools [ OR
= 1.62 ( 95% CI, 1.20–2.19) ] but
no association with increasing hours of lifetime pool use (Villanueva et al. 2007). There was no significant difference in risk for ever swimming in pools with any of the genetic polymorphisms under evaluation here. The weak statistical interactions observed with GSTT1 present (vs

. GSTT1 null) and with GSTZ1 CT/TT ( vs

. CC) were in a direction contrary to expec

tation, given what we observed for interac

tions with THMs [see Supplemental Material, Table 2A,B (doi:10. 1289/ehp.1002206)].



### Discussion DBPs were previously found to be a blad

der cancer risk factor in this case

– control study in Spain, as well as in other settings (Villanueva et al. 2004, 2007). In the present study we found significant differences in the dose

– response relation of bladder cancer risk with increasing average long-term exposure to DBPs (as represented by THMs) among sub

jects with differing genotypes in each of three candidate genes. GSTT1 and GSTZ1 code for enzymes in the respective biotransformation pathways of two groups of DBPs, the bromi

nated THMs and the α -haloacids. CYP2E1 oxidizes THMs and likely many other com

pounds in the DBP mixture. Overall, without considering DBP effects or interaction, we found a weak, non significant overall associa

tion between poly morphisms in each of these genes and bladder cancer risk. Associations between increasing quartiles of THMs and bladder cancer were stronger among subjects with GSTT1 +/+ or +/– versus –/–, GSTZ1 rs1046428 CT or TT versus CC, and CYP2E1 rs2031920 CC versus CT (no subjects had TT), with statistically significant inter

actions for each of the respective gene vari

ants. Among the 195 cases and 192 controls with GSTT1 present and GSTZ1 rs1046428 CT/TT, ORs (95% CIs) for quartiles 2, 3, and 4 of long-term average THMs were 1.5 (0.7–3.5), 3.4 (1.4–8.2), and 5.9 (1.8–19.0), respectively, relative to quartile 1. We also found that main effect estimates for bladder cancer in association with these polymor

phisms varied by THM level, with significant associations for all three high-risk genotypes among subjects within the highest quartile of long-term THMs (Table 4). The super

multiplicative inter actions between residen

tial water THM level and polymorphisms in GSTT1

, GSTZ1 , and CYP2E1 are consistent with the hypothe sis that these genes influence the metabolism of carcinogens in the DBP mixture in drinking water. GSTZ1, conserved over a long evolu

tionary period, plays a key role in the catabo

lism of phenyl alanine and tyrosine. In addition, GSTZ1 transforms several xeno

biotic α -haloacid substrates. The HAAs dichloroacetic acid, bromo chloro acetic acid, and dibromo acetic acid are transformed to glyoxylic acid, and 2,2-dichloro propanoic acid is metabolized to pyruvic acid (Board and Anders 2005). In rodents, dichloro acetic acid causes liver cancer and dibromo acetic acid is a multi site carcinogen (DeAngelo et al. 1999; Melnick et al. 2007). Among GSTZ-depleted rats, total body clearance of dihaloacetic acids

was 3–10 times lower than in rats with nor

mal levels of GSTzeta (Saghir and Schultz 2005). Several SNPs in the GSTZ1 gene are known (Blackburn et al. 2001). Notably, the rs1046428 T allele has been observed to have low enzymatic activity for transformation of dichloro acetic acid compared with the C allele (Blackburn et al. 2001), consistent with our finding of elevated risk for individuals with this genotype. Many other α

- haloacids in the DBP mixture that have not been tested for carcinogenicity may also serve as substrates for GSTZ1 and thereby participate in the interaction we observed. Haplotype analysis for GSTZ1 did not reveal notable elevated or lowered risk of bladder cancer. A common polymorphic variant of GSTT1 is the null form of the allele, which is associated with lack of enzymatic activity. About 20% of Caucasians are homo zygous null for this gene (Raimondi et al. 2006). In our control population, 22.5% were homo

zygous null. Brominated THMs are muta

genic and carcinogenic and are among the **Table 3.** OR s (95% CI s ) for the combined effects of polymorphic forms of GSTT1 and GSTZ1

. a

GSTT1 null and GSTZ1 CC GSTT1 present and GSTZ1 CT/TT Average THM n (cases/controls) OR (95% CI) n (cases/controls) OR (95% CI) p interaction b

≤ 8.0 19/17 1.0 (reference) 40/51 1.0 (reference)

> 8.0–26.0 25/24 1.1 (0.4–3.0) 35/45 1.5 (0.7–3.5) 26.0–49.0 21/29 1.1 (0.4–3.1) 61/49 3.4 (1.4–8.2)

> 49.0 17/26 1.5 (0.4–5.4) 59/47 5.9 (1.8–19.0)
p trend 0.57 0.0012 0.0052

a OR (95% CI) from logistic regression adjusted for age (continuous), sex, smoking status (never/former/current), size of the municipality of longest residence until 18 years of age , education (three strata), geographic area ( six strata), and overall quality of interview. Analysis was r estricted to subjects with either both GSTT1 null and GSTZ1 rs1046428 CC or both GSTT1 present and GSTZ1 rs1046428 CT/TT. b p -Value for multiplicative interaction between the average THM level (age 15 to index year) and the indicated genetic characteristic; the calculation uses the median level of average THM within each successive quartile.

**Table 4.** OR s and 95% CI s for five polymorphic genes ( GSTT1

, GSTZ1

, CYP2E1

, NAT2

, GSTM1 ) within each of four strata of average level of THM in drinking water, in the period from 15 years of age to the index year. a

Average THM (μg/L) GSTT1 present vs. null GSTZ1 rs1046428, CT/TT vs. CC CYP2E1 rs2031920, CC vs. CT/TT NAT2 , slow vs. rapid/intermediate GSTM1 , null vs. present

≤ 8.0 0.9 (0.5–1.5) 0.7 (0.4–1.2) 0.8 (0.5–1.5) 1.3 (0.8–2.0) 1.8 (1.1–2.9)

> 8.0–26.0 0.9 (0.5–1.6) 1.0 (0.6–1.6) 1.2 (0.7–2.0) 1.5 (0.9–2.5) 1.6 (1.0–2.5)

> 26.0–49.0 1.5 (0.9–2.5) 1.0 (0.6–1.6) 1.9 (1.1–3.3) 1.1 (0.7–1.8) 1.8 (1.2–2.8)

> 49.0 1.8 (1.1–3.1) 1.6 (1.0–2.6) 2.0 (1.1–3.9) 1.4 (0.9–2.2) 1.9 (1.2–2.9)

a The referent (OR = 1.0) for each cell in the table, specific to a THM level, is indicated in each column heading. OR (95% CI) from logistic regression adjusted for age (continuous), sex, smoking status (never/former/current), size of the munici

pality of longest residence until 18 years of age , education (three strata), geographic area ( six strata), and overall quality of interview.Bladder cancer, DBPs, and genetic polymorphisms

Environmental Health Perspectives • volume 118 | number 11 | November 2010 

### 1549 most prevalent DBPs in chlorinated drinking water. Pegram et al. (1997) and DeMarini

et al. (1997) demon strated that in Salmonella transfected with rat GSTT1‐1+ , brominated THMs are activated to mutagens, and they identified the specific mutagenic transitions (GC → AT) involved. Dibromonitromethane, a DBP that has not been tested for carcino

genicity, is also activated to a mutagen by a transgenic strain of Salmonella expressing GSTT1-1 (Kundu et al. 2004). Authors of both in vivo and in vitro studies have sug

gested a model whereby brominated THMs, after dermal absorption and inhalation, escape first-pass hepatic metabolism and reach target tissues in the urinary tract where the relative proportion of GSTT1 and oxidative enzymes is more favorable for GSTT1-mediated metabo lism (Landi et al. 1999; Ross and Pegram 2003

, 2004). In the bladder, the bro

minated THM could be activated to mutagens
in a GSTT1+ person, leading to increased bladder cancer risk. Findings from experimen

tal exposures to humans are also consistent with our observations. Exposure of humans to BDCM showed that 2 of 10 subjects, each of whom were GSTT1 null and had low CYP2E1 activity, had the lowest total BDCM metabolism (i.e

. , the highest blood levels of

un metabolized BDCM) and the highest peak levels of mutagenicity in their urine because of un metabolized urinary BDCM (Leavens

et al. 2007). In that experimental study, der

mal exposure to BDCM resulted in blood levels 25–130 times higher than those from oral exposure, indicating that this is a major route of exposure to brominated THM. This is consistent with elevated risk for bladder cancer via showering, bathing, and/or swim

ming (Villanueva et al. 2007). Supporting ourobservation of a positive interaction of longterm DBPs with GSTT1+ is a finding of low risk among populations with the GSTT1 null genotype from an analysis of international data (Kim et al. 2002). Renal cell carcinoma risk is elevated among GSTT1+ individuals with occupational exposure to pesticides or to solvents such as trichloro ethylene, possibly through a similar mechanism (Buzio et al. 2003; Karami et al. 2008). The phase I metabolic enzyme CYP2E1 oxidizes a wide variety of alkanes, alkenes, and aromatic and halogenated hydrocarbons and activates many of them to carcinogenic com

pounds (Bolt et al. 2003). Several chemical species in the DBP mixture are potential substrates of CYP2E1. Our data, revealing a signifi cant interaction between the level of long-term DBP exposure and the –1054C>T (sometimes designated as –1053C>T) rs2031920 SNP of CYP2E1 , suggests the possi

bility of carcinogenic activation of one or more constituents of the DBP mixture by the com

mon form of this enzyme. However, we make this observation cautiously. Although CYP2E1 levels are partially determined by genetic fac

tors, the enzyme is highly inducible by alco

hol and other factors

, and its synthesis can be inhibited by food constituents (Oneta et al. 2002; Perocco et al. 2006). We were un able to control for these latter effects. The relatively small number of subjects with the rs2031920 CT/TT genotype (37 cases, 42 controls) con

tributed to the relative instability of this find

ing. In addition, our observation of an OR

< 1.0 among the most highly exposed subjects with this genotype suggests a cautious interpre

tation of the CYP2E1 findings. It is unlikely that the aromatic amine sub

strates for NAT2, found in tobacco products and linked with bladder cancer, are pres

ent in the DBP mixture (Richardson 2003). However, DBP mixtures are complex

, and we felt it worthwhile to evaluate the possi

bility of an interaction between DBPs and slow/rapid acetylation genotypes of NAT2 in risk of bladder cancer. Our expectation of little or no interaction was borne out in the data. The mechanism underlying associations between bladder cancer and the GSTM1 -nullgenotype is not yet well understood (GarciaClosas et al. 2005). Our data indicated that the strength of the association of bladder cancer with GSTM1 null is not affected by exposure to DBPs. As our primary metric of exposure to DBPs, we used the long-term average THM concentration at the household level (Villanueva et al. 2007). DBPs are a complex mixture of halogenated organics whose com

position and concentration vary in time and space. Hundreds of individual chemical spe

cies have been identified (Richardson 2003). THMs and HAAs are the chemical groups found at the highest concentrations in most mixtures, typically accounting for 20–30% of bound halogen. Although there is variabil

ity among the chemical components of DBP mixtures, the correlation coefficient between THMs and HAAs is usually > 0.7, and THM levels have been used in many epidemiologic studies as a surrogate for the full mixture. Although THMs and halo acetic acids are the most common chemical species within the DBP mixture, they may not be the most toxic/ carcinogenic, and one or more of the poly

morphisms of interest may be acting in impor

tant ways on other compounds with levels that are correlated with THMs. Elevated levels of THMs and other DBPs are common in chlorinated swimming pools and cause elevated THM blood levels among swimmers (Aggazzotti et al. 1998). We found no significant interaction of the poly morphisms studied here with ever use of swimming pools. However, the number of pool users was small, findings were not statistically stable, and the relative concentration s of various DBPs in pools differ from those in drinking water, precluding interpretation of these findings. In this hospital-based study, we had a high response rate among both cases and controls, and all diagnoses were histologically con

firmed. Controls were matched to cases on age group, sex, and geographic area of residence at diagnosis. Although matching on area of residence may result in over matching for type of water, possibly biasing relative risks toward the null, most hospital catchment areas were large enough to provide substantial variabil

ity in water sources. We examined residential history since 15 years of age, so that persons who may have had the same water source at the time of the study could easily have lived in other places previously. We conducted this analysis on a subset of the full study popula

tion, namely

, subjects with genotype data and reliable information on DBP exposures for at least 70% of years between 15 years of age and diagnosis (cases) or interview (controls). We included 680 cases (of 1,219 in the full study) and 714 controls (of 1,271), and associations between bladder cancer and cigarette smoking, long-term exposure to THMs, and the main effects of NAT2 and GSTM1 (Garcia-Closas

et al. 2005; Samanic et al. 2006; Villanueva

et al. 2007) were comparable with those esti

mated for the full study population, suggesting that this subgroup is comparable with the full set of patients recruited to the study. Bladder cancer risk has been consistently linked with long-term exposure to DBPs, a complex mixture of halogenated compounds in drinking water created when chlorine is used as a disinfectant (Cantor et al. 1998; King and Marrett 1996; McGeehin et al. 1993; Villanueva et al. 2004). Experimental evidence of mutagenicity and carcinogenicity supports these findings (Komulainen 2004; Richardson

et al. 2007). Prior to this study, evidence was considered inadequate to firmly support a causal role for DBPs in the etiology of blad

der cancer, primarily for two reasons . First, relative risks reported by most epidemiologic studies have been modest, with OR s typically

< 2 , posing the possibility that observed asso

ciations may be due to bias

. Second, there have been inconsistencies in sex-specific findings. The evidence of elevated bladder cancer risk from the present study, based on inter action of DBPs with candidate genes known to be in the biotransformation pathways of known carcino

gens and mutagens in the mixture, strengthens the case for a causal interpretation. Because

> 20% of the controls in our study population were joint carriers of the high- risk genotypes of the three genes we evaluated, our findings may have significant public health implications in relation to preventing cancers following expo

sure to these water contaminants. Early replica

tion of these findings in another population is warranted.Cantor et al.



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### Appendix I: Participating Study Centers in Spain Institut Municipal d’Investigacio Medica,

Universitat Pompeu Fabra, Barcelona Hospital del Mar, Universitat Autonoma

de Barcelona, Barcelona Hospital Germans Trias i Pujol, Badalona,

Barcelona Hospital de Sant Boi, Sant Boi de Llobregat,

Barcelona Consorci Hospitalari Parc Tauli, Sabadell Centre Hospitalari i Cardiologic, Manresa,

Barcelona Hospital Universitario de Canarias,

La Laguna, Tenerife Hospital Universitario Nuestra Senora de la

Candelaria, Tenerife Hospital General Universitario de Elche,

Universidad Miguel Hernandez, Elche, Alicante Universidad de Oviedo, Oviedo, Asturias Hospital San Agustin, Aviles, Asturias Hospital Central Covadonga, Oviedo, Asturias Hospital Central General, Oviedo, Asturias Hospital de Cabuenes, Gijon, Asturias Hospital de Jove, Gijon, Asturias Hospital de Cruz Roja, Gijon, Asturias Hospital Alvarez-Buylla

, Mieres, Asturias Hospital Jarrio, Coana, Asturias Hospital Carmen y Severo Ochoa, Cangas,

Asturias