Nitrate is a natural compound of green vegetables, such as lettuce and spinach, and root

vegetables, such as beets. Nitrate is also present in drinking water (Gangolli et al. 1994;

McKnight et al. 1999; van Loon et al. 1997,

1998). In the late 1990s, the concentration of

nitrate in the Netherlands increased in vegetables and drinking water due to cultivation

and the use of artificial fertilizers (van Loon

et al. 1998), and these nitrate concentrations

have remained stable to date (Versteegh et al.

2004). This continued high concentration

causes growing concern because of the potential health risks of the metabolites of nitrate

and because of their potential relationship

with cancer.

There is still a relative deficit of epidemiologic data addressing the association between

nitrate exposure and cancer risk. Most of the

epidemiologic studies that are available have

focused on gastric cancer risk (Boeing et al.

1991; Buiatti et al. 1990; Cantor 1997;

Forman 1989; Hansson et al. 1994; Risch

et al. 1985; van Loon et al. 1997), but showing little support for the supposed relationship

between nitrate and gastric cancer risk.

However, an association between nitrate exposure and bladder cancer risk is biologically

plausible.

After ingestion, approximately 20% of

nitrate is endogenously transformed to nitrite

by the bacterial flora of the oral cavity (Weyer

et al. 2001). Nitrite can react in the stomach

with foodborne secondary amines or amides

to form N-nitroso compounds (NOCs),

depending on the availability of nitrate in the

stomach (Mirvish et al. 1987; van Loon et al.

1998; Walker 1990; Weyer et al. 2001).

Because approximately 70% of the orally

ingested nitrate is excreted in the urine, nitrosation may also occur in the bladder (Gulis

et al. 2002; Preston-Martin and Correa 1989;

Weyer et al. 2001). Several lifestyle-related

factors can influence nitrosation. Food components, such as vitamins C and E, may

inhibit the conversion of nitrate into nitrite or

block nitrosation (Council of Europe 1995),

whereas other factors, such as smoking cigarettes, can promote nitrosation (Ward et al.

2003; Weyer et al. 2001). Smokers appear to

have a lower concentration of nitrate in the

blood because of the higher thiocyanate concentration, which can be up to four times

higher than in nonsmokers (Council of

Europe 1995; Mirvish et al. 1987). It is not

nitrate per se but its metabolites that are

potent rodent carcinogens (Tricker and

Preussmann 1991), inducing several types of

cancer including cancer of the stomach, colon,

and lymphatic and hematopoietic system, and

possibly bladder (Bogovski and Bogovski

1981; Gulis et al. 2002; Mirvish et al. 1987). 

Previous epidemiologic studies focused on

nitrate exposure and bladder cancer risk are

sparse; also, they have addressed nitrate intake

only from drinking water and have used ecologic (Gulis et al. 2002; Morales-Suarez-Varela

et al. 1995) or case–control (Ward et al. 2003)

designs. Only one prospective study on drinking-water nitrate intake and bladder cancer risk

has been published so far (Weyer et al. 2001).

In that study of the prospective Iowa Women’s

Health Study, Weyer et al. (2001) found an

increased risk of bladder cancer among older

women exposed to relatively low drinkingwater levels of > 2.46 mg/L/day nitrate, compared with women using < 2.46 mg/L/day

[incidence rate ratios (RR), 2.83; 95% confidence interval (CI), 1.11–7.19]. Weyer et al.

(2001), however, could not provide data on

men (Kantoff 1992).

In the present study, the prospective

design, the possibility of investigating nitrate in

both food and drinking water, the large study

population including both men and women,

and the possibility of studying confounding

and effect modification by environmental

characteristics allowed us to study the association between nitrate intake and bladder cancer

incidence in more detail in the Netherlands

Cohort Study.

Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1527

Research

Address correspondence to M.P. Zeegers, Unit of

Genetic Epidemiology, Department of Public Health

and Epidemiology, University of Birmingham, Public

Health Building, Edgbaston, Birmingham, B15 2TT,

United Kingdom. Telephone: 44 (121) 414-7878.

Fax: 44 (121) 414-7878. E-mail: m.p.zeegers@

bham.ac.uk

We thank the participants, the cancer registries (IKA,

IKL, IKMN, IKN, IKO, IKR, IKST, IKW, IKZ and

VIKC), and the Netherlands nationwide registry of

pathology (PALGA). We also thank A. Volovics and

A. Kester for statistical advice; L. Schouten, S. van de

Crommert, H. Brants, J. Nelissen, C. de Zwart,

M. Moll, W. van Dijk, M. Jansen, and A. Pisters for

assistance; and H. van Montfort, T. van Moergastel,

L. van den Bosch, and R. Schmeitz for programming

assistance.

The Netherlands Cohort Study was supported in

part by the Dutch Cancer Society. M.P.Z. is partly

funded by Cancer Research UK.

The authors declare they have no competing

financial interests.

Received 14 February 2006; accepted 13 July 2006.

Nitrate Intake Does Not Influence Bladder Cancer Risk: The Netherlands

Cohort Study

Maurice P. Zeegers,1,2,3 Roel F.M. Selen,3 Jos C.S. Kleinjans,4 R. Alexandra Goldbohm,5 and Piet A. van den Brandt3

1Department of Public Health and Epidemiology, University of Birmingham, Birmingham, United Kingdom; 2Comprehensive Cancer

Institute Limburg, Department of General Practice, Catholic University of Leuven, Leuven, Belgium; 3Department of Epidemiology, and 4Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, the Netherlands; 5Department of Food and

Chemical Risk Analysis, TNO Quality of Life, Zeist, the Netherlands

OBJECTIVES: N-nitroso compounds, endogenously formed from nitrate-derived nitrite, are suspected

to be important bladder carcinogens. However, the association between nitrate exposure from food or

drinking water and bladder cancer has not been substantially investigated in epidemiologic studies.

METHODS: We evaluated the associations between nitrate exposure and bladder cancer in the

Netherlands Cohort Study, conducted among 120,852 men and women, 55–69 years of age at

entry. Information on nitrate from diet was collected via a food frequency questionnaire in 1986

and a database on nitrate content of foods. Individual nitrate exposures from beverages prepared

with tap water were calculated by linking the postal code of individual residence at baseline to

water company data. After 9.3 years of follow-up and after excluding subjects with incomplete or

inconsistent dietary data, 889 cases and 4,441 subcohort members were available for multivariate

analyses. We calculated incidence rate ratios (RR) and corresponding 95% confidence intervals

(CIs) using Cox regression analyses. We also evaluated possible effect modification of dietary intake

of vitamins C and E (low/high) and cigarette smoking (never/ever).

RESULTS: The multivariate RRs for nitrate exposure from food, drinking water, and estimated total

nitrate exposure were 1.06 (95% CI, 0.81–1.31), 1.06 (95% CI, 0.82–1.37), and 1.09 (95% CI,

0.84–1.42), respectively, comparing the highest to the lowest quintiles of intake. Dietary intake of

vitamins C and E (low/high) and cigarette smoking (never/ever) had no significant impact on these

results.

CONCLUSION: Although the association between nitrate exposure and bladder cancer risk is biologically plausible, our results in this study do not support an association between nitrate exposure and

bladder cancer risk.

KEY WORDS: bladder cancer, cohort study, epidemiology, etiology, nitrate. Environ Health Perspect

114:1527–1531 (2006). doi:10.1289/ehp.9098 available via http://dx.doi.org/ [Online 13 July 2006]
Materials and Methods

Study population. The design of the

Netherlands Cohort Study (NLCS) has been

reported in detail elsewhere (van den Brandt

et al. 1990). Briefly, the NLCS was initiated in

September 1986 and includes 58,279 men and

62,573 women 55–69 years of age at baseline.

Ethical approval was obtained from the

University Hospital Maastricht (Maastricht,

the Netherlands) and TNO. This study population originated from 204 municipal registries

throughout the country. All cohort members

consented to participation by completing a

mailed, self-administered questionnaire on risk

factors for cancer.

For reasons of efficiency in data processing

and analysis, we used the case–cohort approach

(Prentice 1986). Cases were derived from the

entire cohort (providing numerator information for calculation of cancer incidence rates),

and accumulated person-years at risk in the

total cohort were estimated using a random

subcohort sample (providing denominator

information for the rates). The subcohort was

sampled directly after identification of all

cohort members and has been followed up

biennially for vital status information.

Follow-up. The follow-up for incident

bladder cancer in the entire cohort was established by computerized record linkage with

the cancer registries in the Netherlands and

the Dutch national database of pathology

reports (PALGA). Completeness of follow-up

of cancer has been estimated to be at least

96% (Goldbohm et al. 1994). The analysis is

restricted to cancer incidence in 9.3 years of

follow up, from September 1986 through

December 1995. During this follow-up,

955 bladder cancer cases were registered.

Nitrate intake. Each participant’s usual consumption of food and beverages during the year

preceding the start of the study was assessed at

baseline with a 150-item semiquantitative food

frequency questionnaire specifically developed

to measure nutrient contents such as nitrate.

This questionnaire has been validated against a

9-day diet record (Goldbohm et al. 1994).

Food composition values for nitrate were

derived from the databank on contaminants in

food from the State Institute for Quality

Control of Agricultural Products (RIKILT;

Wageningen, the Netherlands) (van Loon 1997

et al. 1998). Estimations were based on the

mean nitrate contents between 1985 and 1989,

in which distinction for some vegetables (e.g.,

endive, lettuce) was made between summer and

winter. Furthermore, information on nitrate

losses during preparation (washing, cutting,

cooking) were considered. For several vegetables

(endive, spinach, cabbage) and for potatoes,

experimental data were available regarding

nitrate losses during preparation (Driessen

1989; van de Worp 1987). For other vegetables, nitrate losses were estimated to be 40%.

For water, we combined information about

nitrate (NO3

–/L) content in drinking water

from all 364 pumping stations in the

Netherlands in 1986 [Vereniging van

Exploitanten van Waterleiding bedrijven in

Nederland (VEWIN) 1989]. In this way, we

could determine the nitrate concentration in

drinking water for each home address by

postal code. If more pumping stations delivered drinking water to a single residence, a

weighted mean was calculated. To calculate

the nitrate intake from water, we used the

information about the amount of water, coffee, tea, and soup consumed, which where

derived from the questionnaire.

Statistical analysis. Subjects with incomplete or inconsistent dietary data were excluded

from the analyses (Goldbohm et al. 1994), leaving 889 cases and 4,441 subcohort members

available for analyses. Because of additional

missing values for chemical water quality,

871 cases and 4,359 subcohort members were

available for analyses on nitrate exposure from

drinking water and total nitrate exposure.

Incidence rate ratios (RRs) and corresponding 95% confidence intervals (CIs) for

bladder cancer were estimated in age- and sexadjusted and multivariable case–cohort analyses using the Cox proportional hazards model

(Cox 1972) processed with the Stata statistical

software package (Cleves et al. 2002). We

estimated SEs using the robust Huber-White

sandwich estimator to account for additional

variance introduced by sampling from the

cohort (Lin and Wei 1989). The proportional

hazards assumption was tested using the

scaled Schoenfeld residuals (Schoenfeld

1982). Tests for dose–response trends in risk

of bladder cancer were assessed by fitting

ordinal exposure variables as continuous

terms. All p-values presented are two-sided.

Statistical tests for interaction were based on

Wald statistics of interaction product terms.

The following variables have been considered as potential confounding factors, based

on earlier analyses (Zeegers et al. 2001a,

2001b, 2001c, 2001d, 2002a, 2002b): age

(years), alcohol consumption (grams per day),

coffee consumption (cups per day), tea consumption (cups per day), water consumption

(liters per day), vegetable consumption (grams

per day), fruit consumption (grams per day),

dietary vitamin C and E intake (milligrams

per day), cigarette smoking (never/ever), current cigarette smoking (percentage yes),

smoking amount (cigarettes per day), smoking duration (years of cigarette smoking), first

degree family history of bladder cancer (percentage yes), and high-risk occupational exposure to dye, rubber, leather or vehicle fumes

(percentage ever). Furthermore, other variables were tested as possible effect modifiers

between nitrate exposure from diet and drinking water (low/high exposure) and bladder

cancer risk: intake of dietary vitamins C and

E (low/high) and smoking of cigarettes

(never/ever). Noninvasive tumors [carcinoma

in situ (Tis), noninvasive papillary carcinoma

(Ta), and submucosal invasion (T1)] were

evaluated separately from invasive tumors

[invasion of muscle (T2)–invasion of prostate,

uterus, vagina, pelvic wall, or abdominal wall

(T4)] to test whether bladder cancer progression is associated with nitrate intake.

Results

Table 1 presents the average baseline nitrate

levels from food and drinking water and the

Zeegers et al.

1528 VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives

Table 1. Description of mean daily intake of nitrate in food and drinking water and estimated total nitrate

intake, as well as the distribution of potential confounding factors: the Netherlands Cohort Study,

1986–1995.

Exposure variable Cases Subcohort

Nitrate from food (mg/day) 104.5 ± 43.4 104.5 ± 44.0

Nitrate from drinking water (mg/day) 5.3 ± 6.2 4.9 ± 6.2

Total nitrate intake 109.8 ± 44.3 109.4 ± 45.2

Potential risk factors

Age (years) 62.5 ± 4.1 61.4 ± 4.2

Alcohol intake (g/day) 15.8 ± 17.9 10.4 ± 14.4

Coffee consumption (cups/day) 5.9 ± 3.0 5.4 ± 2.7

Tea consumption (cups/day) 3.0 ± 2.5 3.5 ± 2.5

Water consumption (L/day) 2.1 ± 0.5 2.1 ± 0.5

Total vegetable consumption (g/day) 190.8 ± 79.4 193.4 ± 83.0

Total fruit consumption (g/day) 154.3 ± 122.4 175.5 ± 119.5

Vitamin C intake (mg/day) 98.7 ± 43.8 103.3 ± 43.8

Vitamin E intake (mg/day) 14.1 ± 6.3 13.4 ± 6.2

Smoking amount (cigarettes/day)a 17.8 ± 11.1 15.2 ± 10.2

Smoking duration (years)a 37.1 ± 11.6 31.7 ± 12.3

Sex (% male)b 766 (86.2) 2,166 (49.1)

Cigarette smoking (% ever)b 780 (87.7) 2,827 (64.1)

Current cigarette smoking (% yes)b 393 (44.2) 1,250 (28.1)

Family history of bladder cancer (% yes)b 10 (1.1) 85 (1.9)

High risk occupation (% yes)b 7 (0.8) 17 (0.4)

Values shown are mean ± SD, except where indicated.

aAmong ever smokers only. bValues shown are number (%).
estimated total nitrate intake per day among

bladder cancer cases and the subcohort members. Although exposure to nitrate from food

was comparable between cases and subcohort

members, nitrate exposure from drinking

water and estimated total nitrate intake

appeared to be somewhat higher among cases.

Of the potential confounders, age, alcohol

intake, coffee consumption, water consumption, dietary vitamin E intake, cigarette smoking amount, and smoking duration were

somewhat higher among cases than among

subcohort members, whereas tea consumption, dietary vitamin C intake, and fruit and

vegetable consumption were higher among

subcohort members. Most differences, however, were nonsignificant. As expected, men

were overrepresented among cases (86.2%),

whereas the male/female distribution was

about equal among subcohort members.

Most of the cases (87.8%) had smoked cigarettes, compared to only 64.1% of the subcohort members. This contrast was stronger

for current smoking at baseline, when 44.2%

of the cases and 28.2% of the subcohort

members smoked. The frequency of having

first degree family members with bladder cancer or having worked in a high risk occupation were very low among both cases and

subcohort members (Table 1).

Cox regression analyses showed that only

age, sex, and cigarette smoking were confounders; therefore, we adjusted for these in

subsequent multivariate analyses. The RRs for

nitrate exposure from food, drinking water,

and estimated total nitrate exposure are presented in Table 2. When adjusted for age and

sex, the RR for nitrate exposure from food was

1.01 (95% CI, 0.79–1.29; p-trend = 0.72),

comparing the highest quintile with the lowest

quintile. After adjustment for cigarette smoking, the RR increased slightly to 1.06 (95% CI,

0.81–1.37; p-trend = 0.96). Additional adjustment for nitrate from drinking water did not

change these estimates substantially. When

comparing the highest quintile with the lowest

quintile of nitrate exposure from drinking

water, the RR was 1.11 (95% CI, 0.87–1.41;

p-trend = 0.14), after adjusting for age and sex.

When adjusted for age, sex and smoking, the

RR decreased to 1.06 (95% CI, 0.82–1.37;

p-trend = 0.23). This RR did not change further after additional adjustment for nitrate

exposure from food. The age- and sex-adjusted

RR for estimated total nitrate exposure was

1.03 (95% CI, 0.80–1.32; p-trend = 0.86),

when comparing the highest quintile with the

lowest quintile of exposure. When adjusted for

age, sex and smoking, this RR increased to

1.06 (95% CI, 0.82–1.38; p-trend = 0.77).

These results were similar for men and women

for nitrate from food (p = 0.62), nitrate from

water (p = 0.14) and total nitrate (p = 0.74)

(data not shown).

Vitamin C (p = 0.63), vitamin E (p = 0.62),

and cigarette smoking (p = 0.11) did not appear

to be significant effect modifiers in the association between nitrate exposure from food and

bladder cancer risk (Table 3). Smoking was

also not an effect modifier when nitrate exposure from drinking water was evaluated

(Table 4).

Table 5 presents the association between

nitrate exposure and transitional cell carcinoma, with respect to tumor invasiveness.

The relative risks for high versus low nitrate

intake from food and/or drinking water did

not differ between invasive and noninvasive

tumors (Table 5).

Discussion

In the present study, we examined the association between nitrate exposure and the risk of

developing bladder cancer in a prospective

cohort study. Although the association

between nitrate exposure and bladder cancer

risk is biologically plausible, our results do not

show a statistically significant association for

baseline nitrate exposure from food or drinking

water or on the basis of total nitrate exposure.

To our knowledge, this is the first study to

investigate nitrate exposure and bladder cancer

risk with respect to tumor invasiveness. In

these analyses, we found no significant results.

The main source of dietary nitrate intake is

vegetables. However, vegetables may contain

certain vitamins that can act as inhibitors of

endogenous nitrosation. As a consequence,

dietary intake of nitrate may not result in a

substantial formation of NOCs (Ward et al.

2003), although conflicting results have been

reported on this issue (Vermeer et al. 1999).

On the other hand, cigarettes also contain

NOCs, which could further promote the

endogenous nitrosation in the body and therefore result in a substantial formation of NOCs.

We have investigated both of these aspects in

this study. Although plausible, interaction

analyses between dietary nitrate intake and

dietary intake of vitamins C and E did not

yield significant results. Although vitamin C

intake decreased the risk of bladder cancer

marginally in this population, high vitamin E

intake did not appear to decrease the risk (data

not shown). Because nitrate from water is often

assumed to pose a greater cancer risk than

nitrate from foods that contain antioxidants

and other protective nutrients, we also evaluated the effects of nitrate-contaminated water

on smokers in a separate analysis. In stratified

analyses, we found that smoking cigarettes did

not significantly change the association

between nitrate intake from either food or

water and bladder cancer risk. However, our

never-smoking stratum was small because of

relatively high European smoking rates. It is

possible that a larger nonsmoking population

would yield a different answer.

A null association for nitrate from drinking water may be explained by the rather low

levels of nitrate in Dutch drinking water. We

assessed the intake of nitrate in drinking water

Nitrate intake and bladder cancer

Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1529

Table 2. RRs (95% CIs) for bladder cancer in relation to quintiles of nitrate exposure in age- and sexadjusted and multivariable analyses: the Netherlands Cohort Study, 1986–1995.

Nitrate exposure Subcohort

[range (median), mg/day] Cases (PY) RR (95% CI)a RR (95% CI)b RR (95% CI)c

Food

2.0–69.0 (57.4) 168 8,512 1.00 (Reference) 1.00 (Reference) 1.00 (Reference)

69.0–88.0 (78.6) 186 8,652 1.14 (0.89–1.45) 1.16 (0.90–1.50) 1.15 (0.89–1.50)

88.0–107.5 (97.8) 181 8,706 1.00 (0.78–1.27) 1.04 (0.80–1.34) 1.05 (0.81–1.37)

107.5–135.3 (119.5) 180 8,707 1.02 (0.80–1.30) 1.06 (0.82–1.37) 1.05 (0.81–1.37)

135.3–451.1 (158.9) 174 8,564 1.01 (0.79–1.29) 1.06 (0.81–1.37) 1.04 (0.80–1.36)

p-Trend 0.72 0.96 0.96

Increment in 10 mg/day 1.00 (0.98–1.01) 1.00 (0.98–1.02) 1.00 (0.98–1.02)

Drinking water

0–0.9 (0.5) 167 8,421 1.00 (Reference) 1.00 (Reference) 1.00 (Reference)

0.9–2.4 (1.4) 147 8,386 0.86 (0.66–1.10) 0.84 (0.64–1.11) 0.84 (0.64–1.11)

2.4–4.4 (3.4) 185 8,476 1.10 (0.86–1.40) 1.11 (0.86–1.44) 1.11 (0.86–1.44)

4.4–7.7 (5.6) 179 8,530 1.05 (0.82–1.34) 1.08 (0.84–1.40) 1.08 (0.84–1.40)

7.7–92.7 (10.6) 193 8,515 1.11 (0.87–1.41) 1.06 (0.82–1.37) 1.06 (0.82–1.38)

p-Trend 0.14 0.23 0.24

Increment in 10 mg/day 1.10 (0.98–1.23) 1.09 (0.96–1.24) 1.09 (0.96–1.24)

Total nitrate

2.4–72.7 (60.5) 159 8,297 1.00 (Reference) 1.00 (Reference)

72.7–92.9 (83.2) 179 8,496 1.20 (0.93–1.53) 1.26 (0.97–1.64)

92.9–112.7 (102.4) 189 8,606 1.11 (0.87–1.42) 1.18 (0.91–1.53)

112.7–140.8 (125.0) 174 8,530 1.08 (0.84–1.39) 1.17 (0.89–1.52)

140.8–525.4 (165.4) 170 8,399 1.03 (0.80–1.32) 1.09 (0.84–1.42)

p-Trend 0.86 0.77

Increment in 10 mg/day 1.00 (0.98–1.02) 1.00 (0.99–1.02)

PY, person-years.

aAdjusted for age (years) and sex. bAdjusted for age (years), sex, current smoking (yes/no), smoking amount

(cigarettes/day), and smoking duration (years). cAdjusted for age (years), sex, current smoking (yes/no), smoking amount

(cigarettes/day), smoking duration (years), and nitrate exposure from food (for drinking water analyses only) or nitrate

exposure from drinking water (for food analyses only).
Zeegers et al.

1530 VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives

from regular drinking water supplies by combining information on nitrate contents in

drinking water from all water companies in

the Netherlands (VEWIN 1989) with information about the distribution for the baseline

year 1986. The average level of nitrate in

Dutch drinking water is 1.68 mg/L (VEWIN

1989). Approximately 5% of the pumping

stations have supplied drinking water with a

nitrate level between 25 and 50 mg/L (van

Duyvenbooden and van Matthysen 1989).

Unfortunately, no information was available

about dietary and water nitrate intake during

follow-up. However, in a reproducibility study,

Goldbohm et al. (1995) demonstrated that the

single food frequency questionnaire measurement could characterize dietary habits for a

period of at least 5 years. Furthermore, data

from the National Institute for Public Health

and the Environment (Bilthoven, the

Netherlands) showed no substantial differences

in average nitrate concentrations in drinking

water within the last decade (Versteegh et al.

2004). Our results are consistent with those of

van Loon et al. (1997) concerning nitrate

intake from food and the lack of an association

between drinking water nitrate intake and cancer: van Loon et al. (1997) found no associations between nitrate intake from Dutch

drinking water and gastric cancer.

Some factors may have influenced the

results of the present study. A possible weakness of this study is that we were not able to

control for bladder infections, a possible risk

factor for bladder cancer. Also, the results

could have been influenced by misclassification

of exposure. Dietary nitrate intake was assessed

by combining information on food intake with

nitrate contents in food, and intake of food

was estimated with a semiquantitative food frequency questionnaire. This method has been

validated against urinary measurement: the

adjusted partial correlation coefficient of intake

verses urinary excretion was 0.59, indicating

that a self-administered dietary questionnaire

can be used to measure usual nitrate intake

(van den Brandt et al. 1989). To account for

the relatively large variation in nitrate levels in

vegetables over seasons, we calculated mean

nitrate contents in vegetables from average

nitrate levels in summer and winter. The

prospective nature of a cohort study, together

with completeness of follow-up, as has been

achieved in this study, reduced the potential

for recall and selection bias to a minimum.

Information bias was also largely avoided

because dietary habits were reported before

bladder cancer was diagnosed.

Our finding of no association with bladder

cancer is consistent with some of the earlier

studies conducted in Slovakia (Gulis et al.

2002) and in Iowa (USA) (Ward et al. 2003).

The first ecologic study, conducted in Slovakia

(Gulis et al. 2002), showed no association

overall; the highest standardized incidence

ratio (1.22; 95% CI, 0.90–1.64) was reported

among people exposed to 0–10 mg/L nitrate

from drinking water. The second ecologic

study, conducted in Spain (Morales-SuarezVarela et al. 1995), reported an association

with bladder cancer among males 55–75 years

of age and exposed to > 50 mg/L of nitrates in

drinking water (RR = 1.4; 95% CI, 0.8–2.48).

A case–control study in Iowa (Ward et al.

2003) found no association with bladder cancer and nitrate levels in water supplies. The

highest quartile odds ratio (OR) for women

exposed to ≥ 2.48 mg/L nitrate in drinking

water was 0.8 (95% CI, 0.4–1.3). The highest

quartile OR for men exposed to ≥ 3.09 mg/L

nitrate in drinking water was 0.5 (95% CI,

0.4–0.8). The Iowa Women’s Health Study

(Weyer et al. 2001) found an increased risk of

bladder cancer among older women exposed

to drinking water levels of nitrate > 2.5 mg/L;

the results showed an RR of 2.83 (95% CI,

1.11–7.19).

We conclude that there is no association

between average nitrate exposure through

food and drinking water, as currently occurs

in the Netherlands, and bladder cancer risk.

However, too few studies have examined this

association. Therefore, further research is

needed in populations exposed to higher

Table 4. RRs (95% CIs) for bladder cancer in relation to nitrate exposure from drinking water, with respect

to cigarette smoking (never/ever): the Netherlands Cohort Study, 1986–1995.

Nitrate intake from drinking water

Low (≤ 3.4 mg/day) High (> 3.4 mg/day)

Cases Subcohort (PY) Cases Subcohort (PY) RR (95% CI)

Cigarette smoking

Never 58 7,692 45 6,764 0.99 (0.64–1.53)a

Ever 352 13,392 416 14,480 1.13 (0.94–1.35)b

PY, person-years.

aAdjusted for age (years), sex, and nitrate exposure from drinking water. bAdjusted for age (years), sex, smoking amount

(cigarettes/day), smoking duration (years), and nitrate exposure from food.

Table 5. RRs (95% CIs) for noninvasive and invasive transitional cell carcinoma of the urinary bladder in

relation to nitrate exposure via food and drinking water and estimated total nitrate exposure: the

Netherlands Cohort Study, 1986–1995.

Noninvasive (Tis, Ta, T1) Invasive (T2–4)

Nitrate Range Subcohort Subcohort 

exposure (mg/day) Cases (PY) RR (95% CI) Cases (PY) RR (95% CI)

Food

Low ≤ 97.9 228 20,557 1.00 (Reference) 246 20,623 1.00 (Reference)

High > 97.9 223 20,644 0.91 (0.73–1.12) 225 20,527 0.91 (0.74–1.12)a

Drinking water

Low ≤ 3.4 214 20,198 1.00 (Reference) 213 20,143 1.00 (Reference)

High > 3.4 230 20,237 1.05 (0.85–1.30) 246 20,227 1.14 (0.92–1.41)a

Total nitrate

Low ≤ 102.4 220 20,157 1.00 (Reference) 234 20,187 1.00 (Reference)

High > 102.4 224 20,278 0.97 (0.79–1.20) 225 20,183 0.96 (0.78–1.19)b

PY, person-years.

aAdjusted for age (years), sex, current cigarette smoking, smoking amount (cigarettes/day), smoking duration (years), and

nitrate exposure from food (for drinking water analyses only) or nitrate exposure from drinking water (for food analyses only).

bAdjusted for age (years), sex, current cigarette smoking, smoking amount (cigarettes/day), and smoking duration (years).

Table 3. RRs (95% CIs) for bladder cancer in relation to nitrate exposure from food, with respect to dietary

vitamin C and E intake (low/high intake) and cigarette smoking (never/ever): the Netherlands Cohort Study,

1986–1995.

Nitrate intake from food

Low (≤ 97.9 mg/day) High (> 97.9 mg/day)

Range Subcohort Subcohort

(mg/day) Cases (PY) Cases (PY) RR (95% CI)

Vitamin C

Low ≤ 96.5 311 14,490 167 7,063 1.00 (0.78–1.26)a

High > 96.5 141 7,075 270 14,513 0.84 (0.65–1.08)a

Vitamin E

Low ≤ 12.2 248 12,679 154 8,629 0.89 (0.69–1.14)a

High > 12.2 204 8,886 283 12,947 1.00 (0.80–1.26)a

Cigarette smoking

Never 64 7,460 45 7,336 0.70 (0.46–1.08)b

Ever 388 14,105 392 14,239 0.98 (0.82–1.17)c

PY, person-years.

aAdjusted for age (years), sex, current smoking, smoking amount (cigarettes/day), smoking duration (years), and nitrate

exposure from drinking water. bAdjusted for age (years), sex, and nitrate exposure from drinking water. cAdjusted for age

(years), sex, smoking amount (cigarettes/day), smoking duration (years), and nitrate exposure from drinking water.
nitrate levels to provide more information

between this potential carcinogen and bladder

cancer risk.

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Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1531