Environmental Health Perspectives • volume 118 | number 11 | November 2010 1545

Research

Chlorine is a cost-effective drinking water disinfectant that has been used since the early 

twentieth century to control a panoply of 

waterborne infectious diseases. By-products of 

the interaction of chlorine with organic precursors 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 disinfection 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 evidence 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 carcinogenicity 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 brominated 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 

carcinogens (DeAngelo et al. 1999; Melnick 

et al. 2007; Tong et al. 1998). Cytochrome 

P450 2E1 (CYP2E1) metabolizes a wide variety of aliphatic hydrocarbons, 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 nonsynonymous 

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 municipalities 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

1Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA; 2KP Cantor Environmental 

LLC, Silver Spring, Maryland, USA; 3Centre for Research in Environmental Epidemiology, Barcelona, Spain; 4Institut Municipal 

d’Investigacio Medica–Hospital del Mar, Barcelona, Spain; 5CIBER Epidemiologia y Salud Publica, Barcelona, Spain; 6Centro Nacional 

de Investigaciones Oncológicas, Madrid, Spain; 7Universidad Pompeu Fabra, Barcelona, Spain; 8Universidad de Oviedo, Oviedo, Spain; 9Unidad de Invesigación, Hospital Universitario de Canarias, La Laguna, Spain; 10Consorci Hospitalari Parc Tauli, Sabadell, Spain; 11Ramon y Cajal University Hospital, Madrid, Spain; 12National School of Public Health, Athens, Greece

Background: Bladder cancer has been linked with long-term exposure to disinfection by-products 

(DBPs) in drinking water.

Objectives: In this study we investigated the combined influence of DBP exposure and polymorphisms 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.

Methods: 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.

Results: 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 (pinteraction

= 0.021), GSTZ1 rs1046428 CT/TT versus CC (pinteraction = 0.018), or CYP2E1 rs2031920 CC 

versus CT/TT (pinteraction = 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.

Conclusions: Polymorphisms in key metabolizing enzymes modified DBP-associated bladder cancer 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.

Key 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 augmented 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 conditions 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% 

ophthalmological disorders, and 3% other 

diseases. Controls were individually matched 

to cases by age at interview within 5-year 

strata, sex, ethnic origin, and hospital catchment 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 sociodemographic 

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 controls (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/bottled/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 questionnaire, we collected historic water quality 

data from approximately 200 local authorities 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 addition, one of us (C.M.V.) measured levels of 

the four THMs [chloroform, bromodichloromethane (BDCM), dibromochloromethane,

and bromoform] 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 concentrations when the water source had not changed. 

When the water source had changed, we calculated the average THM level using the proportion of surface water during the relevant 

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 year 

and 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 exposure (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 mouthwash 

samples as described previously (GarciaClosas et al. 2005). Genotype assays for 

polymorphisms in GSTT1, GSTM1, and 

N-acetyltransferase 2 (NAT2) were conducted 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 elsewhere (Garcia-Closas et al. 2005). GSTT1

genotypes were defined as null (–/–) if a deletion 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 genotypes studied were in Hardy-Weinberg equilibrium 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 (ORs) and 

95% confidence intervals (CIs). ORs were 

adjusted for age (continuous), sex, smoking status (never/former/current), size of the municipality 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, average 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 category for each variable 

included in the models. This applied to a small 

proportion of respondents for each covariate. 

For example, < 1% of respondents were missing 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 modeled as a continuous variable with each quartile 

coded according to its median value.

Interactions between genotypes and exposure to DBPs, as estimated by average THM 

levels serving the household between 15 years 

of age and diagnosis (cases) or interview (controls), were also tested using the likelihood-ratio test to allow estimation of parameters under 

the assumption of genotype–DBP (THMs) 

independence in the source population. 

Tests for multiplicative interaction were used 

to assess whether the genotype ORs within 

categories of DBP exposure or, equivalently, 

DBP ORs within genotype categories differed 

significantly from each other. Haplotype frequencies for GSTZ1 and CYP2E1 were estimated 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 interviewed, 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, nonwhite 

individuals (to limit heterogeneity), and DNA 

quality control difficulties, the study population with adequate genetic material available for analysis numbered 1,150 cases and 
Bladder 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 inadequate exposure data as described above, the 

maximum numbers 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)] smokers of cigarettes in the analysis data set, relative 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) 

(ptrend = 0.029) (Table 1), similar to the findings of Villanueva et al. (2007). These ORs are 

adjusted for the covariates listed above, as are 

all subsequent ORs reported here. Findings 

from Garcia-Closas et al. (2005), from this 

study population, showed significant associations between bladder cancer and NAT2 slow 

acetylator compared with rapid/intermediate 

genotypes [OR (95% CI) of 1.4 (1.2–1.7)]

and GSTM1 null (–/–) versus GSTM1 present genotypes [1.7 (1.4–2.0)]. Estimates in 

the present analyses were similar, with NAT2

(slow compared with rapid plus intermediate 

acetylator type) having an OR (95% CI) of 

1.33 (1.06–1.68), and with GSTM1 null 

compared with the GSTM1 present genotype 

having an OR (95% CI) of 1.77 (1.41–2.22). 

We found slightly elevated, nonsignificant 

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 heterozygotes 

(+/–) and 1.0 (0.8–1.3) for homozygous null 

(–/–) (Garcia-Closas et al. 2005).

We found nonsignificant 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 disequilibrium (pairwise D ́ 

values ranged from 0.98 to 1.0); however,

they were not highly correlated with each 

other (r2 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 disequilibrium (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 revealed no common haplotype 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) (pinteraction

= 0.021), exon 7 +29T>C rs1046428 in 

GSTZ1 (pinteraction = 0.018), and –1054C>T 

rs2031920 in CYP2E1 (pinteraction = 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 significant 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 information beyond the single SNP analysis (data 

not shown). We found no evidence of interaction between either NAT2 slow acetylator 

or GSTM1-null genotype and long-term average THM level (pinteraction = 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 interaction 

between average THM level and combined 

GSTT1 and GSTZ1 rs1046428 genotypes 

(pinteraction = 0.0052) in analyses that included 

Table 1. ORs (95% CIs) 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)

ptrend 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

aNumbers of cases or controls per measure may not 

equal the total number of cases (680) and controls (714) 

because of missing genotype data. bOR (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.

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) pinteractionb

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

ptrend 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

ptrend 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

ptrend 0.33 0.014

aORs (95% CIs) 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. bp-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 (ptrend = 0.0012). 

In contrast, among subjects with both GSTT1

null and GSTZ1 CC genotypes, we found 

no increase in relative risk with increasing THM level (pinteraction = 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 monotonically to 9.3 (2.5–34.0) in the highest quartile 

of THM exposure (> 49.0 μg/L), relative to 

participants with these genotypes in the lowest quartile [≤ 8.0 μg/L; ptrend = 0.0006 with 

pinteraction = 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 expectation, given what we observed for interactions with THMs [see Supplemental Material, 

Table 2A,B (doi:10.1289/ehp.1002206)].

Discussion

DBPs were previously found to be a bladder 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 subjects 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 brominated THMs and the α-haloacids. CYP2E1 

oxidizes THMs and likely many other compounds in the DBP mixture. Overall, without 

considering DBP effects or interaction, we 

found a weak, nonsignificant overall association between polymorphisms 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 interactions for each of the respective gene variants. 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 polymorphisms 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 supermultiplicative interactions between residential water THM level and polymorphisms in 

GSTT1, GSTZ1, and CYP2E1 are consistent 

with the hypothesis that these genes influence 

the metabolism of carcinogens in the DBP 

mixture in drinking water.

GSTZ1, conserved over a long evolutionary period, plays a key role in the catabolism of phenylalanine and tyrosine. In 

addition, GSTZ1 transforms several xenobiotic α-haloacid substrates. The HAAs 

dichloroacetic acid, bromochloroacetic acid, 

and dibromoacetic acid are transformed to 

glyoxylic acid, and 2,2-dichloropropanoic 

acid is metabolized to pyruvic acid (Board and 

Anders 2005). In rodents, dichloroacetic acid 

causes liver cancer and dibromoacetic acid is 

a multisite 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 normal 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 

dichloroacetic 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 homozygous 

null for this gene (Raimondi et al. 2006). In 

our control population, 22.5% were homozygous null. Brominated THMs are mutagenic and carcinogenic and are among the 

Table 3. ORs (95% CIs) 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) pinteractionb

≤ 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)

ptrend 0.57 0.0012 0.0052

aOR (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 restricted to subjects with either both GSTT1 null and GSTZ1 rs1046428 CC or 

both GSTT1 present and GSTZ1 rs1046428 CT/TT. bp-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. ORs and 95% CIs 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)

aThe 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 municipality 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) demonstrated 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 carcinogenicity, 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 suggested 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 

metabolism (Landi et al. 1999; Ross and 

Pegram 2003, 2004). In the bladder, the brominated THM could be activated to mutagens 

in a GSTT1+ person, leading to increased 

bladder cancer risk. Findings from experimental 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 

unmetabolized BDCM) and the highest peak 

levels of mutagenicity in their urine because 

of unmetabolized urinary BDCM (Leavens 

et al. 2007). In that experimental study, dermal 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 swimming (Villanueva et al. 2007). Supporting our 

observation 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 trichloroethylene, 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 compounds (Bolt et al. 2003). Several chemical 

species in the DBP mixture are potential 

substrates of CYP2E1. Our data, revealing 

a significant interaction between the level of 

long-term DBP exposure and the –1054C>T 

(sometimes designated as –1053C>T) 

rs2031920 SNP of CYP2E1, suggests the possibility of carcinogenic activation of one or more 

constituents of the DBP mixture by the common form of this enzyme. However, we make 

this observation cautiously. Although CYP2E1 

levels are partially determined by genetic factors, the enzyme is highly inducible by alcohol and other factors, and its synthesis can be 

inhibited by food constituents (Oneta et al. 

2002; Perocco et al. 2006). We were unable 

to control for these latter effects. The relatively 

small number of subjects with the rs2031920 

CT/TT genotype (37 cases, 42 controls) contributed to the relative instability of this finding. In addition, our observation of an OR

< 1.0 among the most highly exposed subjects 

with this genotype suggests a cautious interpretation of the CYP2E1 findings.

It is unlikely that the aromatic amine substrates for NAT2, found in tobacco products 

and linked with bladder cancer, are present in the DBP mixture (Richardson 2003). 

However, DBP mixtures are complex, and 

we felt it worthwhile to evaluate the possibility 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-null 

genotype 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 composition and concentration vary in time and 

space. Hundreds of individual chemical species 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 variability 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 haloacetic 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 polymorphisms of interest may be acting in important 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 polymorphisms 

studied here with ever use of swimming pools. 

However, the number of pool users was small, 

findings were not statistically stable, and 

the relative concentrations 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 confirmed. 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 overmatching for type 

of water, possibly biasing relative risks toward 

the null, most hospital catchment areas were 

large enough to provide substantial variability 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 population, 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 estimated 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 bladder cancer, primarily for two reasons. First, 

relative risks reported by most epidemiologic 

studies have been modest, with ORs typically 

< 2, posing the possibility that observed associations 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 interaction of 

DBPs with candidate genes known to be in the 

biotransformation pathways of known carcinogens 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 exposure to these water contaminants. Early replication of these findings in another population 

is warranted.
Cantor et al.

1550 volume 118 | number 11 | November 2010 • Environmental Health Perspectives

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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