ORIGINAL ARTICLE

Risk of Non-Hodgkin Lymphoma and Nitrate and Nitrite

From Drinking Water and Diet

Mary H. Ward,* James R. Cerhan,† Joanne S. Colt,* and Patricia Hartge*

Introduction: Nitrate and nitrite are precursors in the in vivo formation of N-nitroso compounds, potent animal carcinogens.

Methods: We conducted a population-based case–control study of

non-Hodgkin lymphoma (NHL) in 1998 to 2000 in Iowa, Detroit,

Seattle, and Los Angeles. Because nitrate levels were elevated in

many drinking water supplies in Iowa, but not in the other study

centers, we evaluated water nitrate levels and risk of NHL in Iowa

only. Monitoring data for public supplies were linked to water

source histories from 1960 onward. Nitrate was measured at interview homes with private wells. We limited most analyses to those

with nitrate estimates for 70% of their person-years since 1960

(181 cases, 142 controls). For those in the diet arm of the study (458

cases, 383 controls from 4 centers) and for Iowa participants in both

the diet and drinking water analyses, we estimated dietary nitrate

and nitrite intake using a 117-item food-frequency questionnaire that

included foods high in nitrate and nitrite. Odds ratios and 95%

confidence intervals were calculated using logistic regression, adjusting for the study matching factors, education, and caloric intake

(diet analyses only).

Results: We found no overall association with the highest quartile of

average drinking water nitrate (2.90 mg/L nitrate-N: odds ratios 

1.2; 95% confidence interval 0.6–2.2) or with years 5 mg/L

(10 years: 1.4; 0.7–2.9). We observed no evidence of an interaction between drinking water nitrate exposure and either vitamin C or

red meat intake, an inhibitor and precursor, respectively, of Nnitroso compound formation. Among those in the diet arm, dietary

nitrate was inversely associated with risk of NHL (highest quartile:

0.54; 0.34–0.86). Dietary nitrite intake was associated with increasing risk (highest quartile: 3.1; 1.7–5.5) largely due to intakes of

bread and cereal sources of nitrite.

Conclusion: Average drinking water nitrate levels below 3 mg/L

were not associated with NHL risk. Our study had limited power to

evaluate higher levels that deserve further study.

(Epidemiology 2006;17: 375–382)

Nitrate has steadily accumulated in our water resourceslargely due to nitrogen fertilizer use, human and animal

waste, and air pollution.1 To protect infants from developing

methemoglobinemia, the U.S. Environmental Protection Agency

set the maximum contaminant level in drinking water of 10

mg/L as nitrate–nitrogen (N) (equivalent to approximately 45

mg/L nitrate).2 This regulation applies only to public water

supplies, not to private wells, and it was not based on estimates

of cancer risk.

Nitrate is a precursor in the formation of N-nitroso

compounds, a class of genotoxic compounds that includes

many animal carcinogens. Endogenous nitrosation is estimated to account for roughly 40% to 75% of the total human

exposure to N-nitroso compounds.3 Other sources of human

exposure include preformed N-nitroso compounds found in

preserved meats and fish, tobacco, and certain occupational

exposures.3 Nitrate intake at the acceptable daily intake level

(3.67 mg/kg body weight, 0.84 mg/kg expressed as nitrate–

nitrogen) results in increased urinary excretion of N-nitroso

compounds, particularly in combination with increased intake

of dietary nitrosatable precursors.4 Dietary compounds that

inhibit endogenous nitrosation include vitamin C and alphatocopherol.

Three ecologic studies5–7 and 3 analytic epidemiologic

studies,8–10 most of which were conducted in areas with

elevated nitrate levels due to agricultural activity, evaluated

exposure to nitrate through public water supplies and risk of

non-Hodgkin lymphoma (NHL). Results are mixed. One

ecologic study6 and one analytic study8 observed an elevated

risk with nitrate levels in public water supplies; no association was observed in the other studies. Two studies8,10 evaluated dietary nitrate, which comes primarily from vegetables,

and found slight inverse associations with higher intake.

We therefore evaluated drinking water and dietary sources

of nitrate as risk factors for NHL in Iowa, an agricultural state

with elevated nitrate levels in many public water supplies. This

analysis was part of a population-based case–control study of

NHL conducted in 4 Surveillance, Epidemiology, and End

Results (SEER) centers to investigate the role of environmental

exposures in NHL risk. We evaluated dietary nitrate and nitrite

intake among cases and controls from all 4 SEER centers who

participated in the diet arm of the study.

METHODS

Study Population

The study design, described in detail elsewhere,11 comprised 4 areas covered by the SEER program of the National

Cancer Institute: the state of Iowa; the Detroit, Michigan

Submitted 27 July 2005; accepted 30 December 2005.

From the *Division of Cancer Epidemiology and Genetics, National Cancer

Institute, NIH/DHHS, Bethesda, MD; and the †Mayo Clinic College of

Medicine, Rochester, MN and the University of Iowa, Iowa City, IA.

Support for data collection was provided by the National Cancer Institute

grant N01-PC-67008. This research was also supported by the Intramural

Research Program of the NIH, National Cancer Institute.

Correspondence: Mary H. Ward, Occupational and Environmental Epidemiology Branch, National Cancer Institute, 6120 Executive Blvd., EPS

8104, Bethesda, MD 20892. E-mail: wardm@mail.nih.gov.

Copyright © 2006 by Lippincott Williams & Wilkins

ISSN: 1044-3983/06/1704-0375

DOI: 10.1097/01.ede.0000219675.79395.9f

Epidemiology • Volume 17, Number 4, July 2006 375
metropolitan area; Los Angeles County, California; and the

Seattle, Washington metropolitan area. We identified as eligible cases newly diagnosed with NHL from 1 July 1998

through 30 June 2000 who were age 20 to 74 years at diagnosis

and not infected with human immunodeficiency virus. All 1,321

newly diagnosed NHL cases were histologically confirmed

and classified into ICD-O-2 based on SEER codes; cases

were collapsed into 4 pathologic groups: follicular, diffuse,

T-cell, and all other classifications.

We selected controls age 20 to 64 years from the

general population of the study areas using random digit

dialing; we selected controls age 65 to 74 years from Medicare eligibility files. In total, 1,057 controls were recruited

and matched to cases by age (5 years), center, race, and

gender. Interviews were conducted in 1998 to 2000.

Drinking Water Analysis

The drinking water analyses were limited to participants of Iowa. A total of 361 (67%) of the eligible cases in

Iowa were interviewed. We selected 478 controls in Iowa and

interviewed 276 (58%) of them. As previously reported,11

case and control participation and response rates were higher

in women than men and higher in whites than other racial

groups. We linked Iowa participants’ and nonparticipants’

current addresses to their census block group and evaluated

the proportion living in an urban area in 2000 and using

public water supplies in 1990 (last year data were available).

We found no substantial differences between participants and

nonparticipants.

Participants were sent a residential calendar in advance

of the in-person interview, which consisted of a table with

separate lines for their year of birth through the interview

year. They were asked to provide their complete address for

every home they lived in from birth to the current year,

indicating the year they moved in and out by a vertical line

between each home; they were also asked to provide information about temporary or summer homes where they lived

for a total of 2 years or longer. Interviewers reviewed the

residential calendar with respondents and probed to obtain

missing information. For each home, respondents were asked

whether the primary drinking water source was a public

supply, a private well, bottled water, or another source.

Nitrate in Public Water Supplies

The Iowa Center for Health Effects of Environmental

Contaminants maintains a database with public water supply

contaminant and treatment data for utilities serving towns

with populations of 400 or more. Data include the specific

sources of the water (surface, groundwater), the years sources

operated, and the estimated percent of the water from each

source by year.12 For smaller utilities, the Iowa Center maintains nitrate data collected under the Safe Drinking Water Act

beginning in the late 1970s. We used nitrate measurement

data for samples taken in the distribution system, which

reflect the water quality at the taps of homes. Multiple

samples taken in the same year were averaged and linked to

an individual’s public water supply history by year and town.

Our exposure assessment was for the period 1960 onward

because nitrate measurements were sparse previously and

because nitrate contamination of public water supplies was

uncommon before the beginning of widespread nitrogen fertilizer use in the 1950s.

For years when a town had no nitrate measurements, an

average level was imputed using a weighted average of the

annual averages in adjacent years. Greater weight was given

to years closer in time to the year with no measurements. The

imputation weights were 1 for 1 to 2 years from a year with

no data, 0.75 for 3 to 4 years, 0.5 for 5 to 6 years, and 0.25

for 7 to 9 years. If there were no data within 10 years, the

nitrate level was considered to be unknown. Levels were

imputed only for the period of years when a city’s sources

and the percent each supplied did not vary by more than 10%.

If a participant reported using bottled water as their primary

drinking water source, we assigned a low nitrate level (0.5 mg

nitrate-N/L). We computed person-years by type of water

source (eg, Iowa private wells, Iowa public supplies, bottled

water) from the year of birth to the year of diagnosis for cases

and to a comparable reference year for controls.

There is no regulatory requirement to monitor nitrate

levels in private wells, and levels can be substantially higher

than public supplies. To reduce misclassification, we limited

most of the nitrate analyses to participants who had public

supplies with an estimated nitrate level for 70% or more of

their person-years since 1960 (181 cases, 142 controls). Among

these cases and controls, the median number of Iowa residences with public water supplies from 1960 onward was 3

(interquartile range IQR 2–4). The person-years with nitrate

measurements from 1960 onward were also similar for cases

and controls. For example, in the 1980s for both cases and

controls, the median number of years with nitrate measurements was 3 (IQR 2–7). In the 1990s, the median years with

measurements was 8 for cases and 7 for controls.

We computed the average nitrate level and the number

of years that an individual had an Iowa public supply with

nitrate levels 5 and 10 mg/L nitrate-N. Our results were

unchanged when we limited our analyses to populations with

more complete water nitrate exposure estimates since 1960

(80% and 90% of person-years); therefore, we only present

results for those with 70% of their person-years with nitrate

estimates.

Nitrate Levels in Private Wells

At the time of the interview, which occurred throughout

the calendar year, we sampled tap water from homes of

participants using a private well as their drinking water

source. The Iowa Hygienic Laboratory measured the combined nitrate and nitrite level using the cadmium reduction

method. Results were obtained for 54 cases and 44 controls in

Iowa. All 54 cases (100%) and 41 of the controls (93%) had

used the well as their primary water source for 5 years or

more. Among this group, we evaluated the nitrate level in

private wells in relation to NHL risk. We also calculated an

average nitrate level and years 5 mg/L that incorporated

both the private well nitrate level and the public supply nitrate

level and limited the analysis to cases and controls for whom

we had nitrate estimates for 70% of their person-years since

1960 (211 cases, 165 controls).

Ward et al Epidemiology • Volume 17, Number 4, July 2006

376 © 2006 Lippincott Williams & Wilkins
Dietary Analysis

Participants from all 4 centers were randomly assigned

to one of 2 study arms, except for black participants who

were assigned to the medical history arm of the study. Each

study arm received a slightly different version of a computerassisted personal interview and different versions of a selfadministered questionnaire (dietary or family medical questionnaire). Among participants assigned to the diet arm, 482

of 552 cases and 417 of 462 controls returned the dietary

questionnaire. Two other cases and 2 other controls inadvertently received and returned the dietary questionnaire for a

total of 484 cases and 419 controls. As previously described,13–15 we excluded subjects from the dietary analyses

if the daily number of reported foods was 4 or 30 for

males and 3 or 30 for females or if the number of food

items missing or left blank exceeded 20% (18 cases and 28

controls), leaving 466 cases and 391 controls. We report

results for dietary nitrate and nitrite intake for all participants

in the diet arm of the study and for diet arm participants who

were also included in the Iowa public water supply analyses

(95 cases, 73 controls).

Dietary and vitamin supplement intake was assessed

using a 117-item food-frequency questionnaire that was a

modified version of the 1995 Revision of the Health Habits

and History Questionnaire.16,17 Modifications included additional questions about tap water intake, meat preparation

methods, and doneness preferences. We added questions

about vegetables containing high levels of nitrate (eg, beets,

celery, radishes) and processed meats that contain nitrite and

N-nitroso compounds. The nitrate and nitrite contents of

foods were determined from the literature, as described previously.18,19 Daily intakes of nitrate and nitrite were calculated by multiplying the frequency of consumption of each

food by the nutrient content of the serving and summing

across all food items. Tap water intake was estimated by

summing reported intake of coffee, tea, and water at home.

Data Analysis

We computed maximum likelihood estimates of the

odds ratios (ORs) and 95% confidence intervals (CIs) using

unconditional logistic regression. We adjusted the ORs for

the study matching factors (age in deciles, gender, race) and

years of education (12 years, 12–15 years, 15 years). The

drinking water analyses were not adjusted for race due to the

small numbers of nonwhite participants in Iowa. We adjusted

for the average population size of participants’ Iowa residences (calculated using 1970, 1980, 1990, and 2000 census

data) if the ORs changed by 20% or more; adjustment for

other study risk factors did not change the results by this

amount and are not presented. Dietary nitrate and nitrite

analyses were also adjusted for total calories and dietary

vitamin C intake as continuous variables. Among those who

participated in the diet arm and were also in the Iowa public

supply analyses, we evaluated the association of drinking

water nitrate and NHL risk stratified by dietary vitamin C

intake, red and processed meat intake, and smoking status,

which are factors known to affect endogenous nitrosation.

RESULTS

Drinking Water Nitrate and Non-Hodgkin

Lymphoma Risk

Cases and controls reported similar histories of using

Iowa public supplies, private wells, and bottled water as their

primary drinking water source (Table 1). As expected, the

Iowa study population overall had used public supplies for

fewer years and private wells for more years than the subgroup in the water nitrate analyses. Duration of private well

use was not associated with NHL risk in either group (not

shown). The majority in both groups had never used bottled

water and had very few years with an unknown water source.

The distribution of cases and controls in the nitrate analysis

by age, sex, and race was not substantially different from the

study population overall. Cases in both groups tended to have

had more years of education than controls. Cases and controls

in the nitrate analyses were less likely to have been farmers

and more likely to have lived in larger towns than the study

population overall, although there were no substantial case/

control differences in farming status or average population of

residences in either group. In both groups, more cases than

controls were in the highest body mass index group (31

kg/m2

). There were fewer nonsmokers among the population

in the drinking water analyses compared with the study

population overall. Intakes of vitamin C and red meat did not

differ substantially between the 2 groups. Likewise, the

distribution of NHL cases by pathologic subgroups was

similar between cases in the drinking water analyses and

cases overall.

The results for years using a public supply with levels 5

mg/L and 10 mg/L nitrate-N are shown in Table 2. Overall,

there was no association with longer duration using supplies

with 5 mg/L. Among men, ORs tended to increase with

increasing duration; among women, there was no association.

Few cases and controls had any years of exposure to a public

supply with nitrate level 10 mg/L; among this group, there was

a slight inverse association with risk of NHL.

The nitrate level averaged over Iowa residences with

public supplies from 1960 through the reference date was

similar between cases and controls (for cases, median 1.4,

IQR 0.7–2.9; for controls, median 1.4, IQR 0.6–2.9).

Increasing quartiles of the average nitrate level were not

associated with risk of NHL (Table 3). The average levels

computed without the last 5 or 10 years of nitrate data were

lower than the average computed over the entire period (for

controls without last 5 years, median 1.4, IQR 0.6–2.7;

without last 10 years, median 1.1, IQR 0.6–2.4). The lagged

average levels were not associated with risk (not shown).

A total of 57% of cases and 61% of controls had no

detectable nitrate in their private well samples, and only 8

cases and 8 controls had nitrate levels that were 5 mg/L or

greater. Compared with those who had no detectable nitrate

(0.2 mg/L), there was no association with nitrate levels in

private wells and NHL risk (for 5 mg/L, OR 1.7, 95%

CI 0.6–5.2; for 5 mg/L, 0.8 2–2.5). When we computed average nitrate levels since 1960 using both public

supply and private well nitrate levels, the distribution was not

Epidemiology • Volume 17, Number 4, July 2006 Non-Hodgkin Lymphoma and Drinking Water Nitrate

© 2006 Lippincott Williams & Wilkins 377
TABLE 1. Characteristics of the Study Population and of the Subgroup Included in the Analyses of Public

Water Supply Nitrate*

Characteristic

Iowa Study Population Subgroup†

Controls

(n  276)

NHL Cases

(n  361)

Controls

(n  142)

NHL Cases

(n  181)

Water sources (years); median (IQR)

Public water supplies in Iowa 30.8 (15.5–44.5) 31.5 (15.5–44.5) 42.5 (35.0–52.0) 41 (33.5–49.5)

Private wells in Iowa 16.2 (0–30.7) 7.0 (0–24.5) 4.0 (0–18.5) 0 (0–12.0)

Bottled water 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0)

Unknown 3.0 (1.0–6.5) 4.0 (1.5–9) 3.0 (1.0–6.5) 3.5 (1.5–8.5)

Tap water intake at home; L/d‡ 1.4 (0.9–2.0) 1.2 (0.6–1.8) 1.4 (1.0–2.0) 1.2 (0.6–2.0)

Age (years); median (IQR) 64.5 (54–70) 62 (50–69) 65.5 (58–70) 64 (49–70)

Sex; %

Male 51 52 51 54

Female 49 48 49 46

Race; %

White only 98 97 98 99

Black 1 1 1 0.5

Other/unknown 1 2 1 0.5

Education (years)

12 10 10 13 10

12–15 71 66 67 69

16 19 24 20 21

Farming status

Never 72 76 83 87

Former 11 8 13 9

Current 17 16 4 3

Average population density of residences

2500 27 26 15 19

2500 to 10,000 24 24 20 22

10 to 50,000 27 25 30 26

50,000 23 25 34 33

Body mass index; kg/m2

20 3 4 3 3

20–25 27 26 27 30

25–30 41 40 41 33

31 24 26 28 32

Cigarette smoking§

Never 45 52 35 50

Former 38 30 45 30

Current 17 18 20 21

Vitamin C intake‡

Less than median (90 mg/d) 54 51 52 54

Median or more 46 49 48 46

Red meat consumption‡

Less than median (18.4 g/d) 54 58 51 55

Median or more 46 42 49 45

NHL pathologic group; %

Follicular 29 30

Diffuse 35 30

T-cell 4 4

Other 32 36

*Percentages do not always total to 100% where there are missing data.

†

Included participants with public water supply nitrate data for 70% of their person-years since 1960.

‡

Tap water, vitamin C, and red meat intakes limited to those with public water supply nitrate data for 70% of their person-years since 1960 and

adequate dietary information. §

Excludes 132 controls and 183 cases in the study population and 65 controls and 84 cases in the subgroup for whom smoking was not ascertained.

Ward et al Epidemiology • Volume 17, Number 4, July 2006

378 © 2006 Lippincott Williams & Wilkins
substantially different from that based on public supply levels

only (Table 3). Among those with 70% of their person years

since 1960 with a known nitrate level in public or private well

supplies, there was no association between the nitrate level

and risk of NHL.

Nitrate intake (in milligrams per day) from public

drinking water supplies was determined for participants in the

water analyses for whom we had information on the usual

amount of tap water consumed at home (95 cases, 73 controls). The median tap water intake at home was 1.2 L/d (IQR

0.6–2.0) among cases and 1.4 L/d (IQR 1.0–2.0) among

controls. The amount of tap water consumed at work was low

(for cases, median 0 L/d, IQR 0–0.35; for controls, median 

0 L/d, IQR 0–0.13). Among controls, nitrate intake from

drinking water (estimated from home intake only) was 2.6

mg/d nitrate-N (IQR 1.2–6.3) among men and 2.4 mg/d (IQR

1.2–5.4) among women. Quartiles of daily nitrate intake from

drinking water showed no association with risk of NHL (not

shown).

We evaluated the association between the average nitrate level in public supplies (3, 3 mg/L) and NHL risk

among those with high (median) and low (median) vitamin C intake (Table 4). The risk of NHL associated with

higher average nitrate levels was elevated among both those

with higher and those with lower vitamin C intakes, but there

was no evidence of an interaction between water nitrate and

vitamin C. Likewise, there was no evidence of an interaction

of drinking water nitrate and red meat intake (not shown).

Compared with participants who had average nitrate levels

3 mg/L and processed meat intake below the median

(18.4 mg/d), risk of NHL was highest among those with 3

mg/L public water supply nitrate and processed meat intake

below the median (OR 3.3, 95% CI 0.9–11.3; 15

cases, 4 controls). There was no elevated risk among those in

the higher category of water nitrate and with processed meat

intake above the median (1.11 0.4–3.2; 11 cases, 9 controls). Stratification by smoking status (current/past; nonsmokers) revealed no evidence of interaction with drinking

water nitrate exposure (not shown).

Dietary Nitrate and Nitrite

Among participants in the diet arm of the study, we

observed an inverse association between dietary nitrate intake

TABLE 2. Association of Years Using an Iowa Public Water

Supply With 5 and 10 mg/L Nitrate-N and NHL*

No. of

Cases

No. of

Controls

OR†

(95% CI)

Years with nitrate-N 5 mg/L

0‡ 82 67 1.0

1–9 64 51 1.0 (0.6–1.7)

10 35 24 1.4 (0.7–2.9)

Men

0‡ 42 38 1.0

1–9 35 22 1.4 (0.7–3.0)

10 21 13 1.7 (0.7–4.3)

Women

0‡ 40 29 1.0

1–9 29 29 0.7 (0.3–1.5)

10 14 11 1.3 (0.5–3.7)

Years with nitrate-N 10 mg/L

0‡ 170 132 1.0

1 11 10 0.6 (0.2–1.5)

*Includes those who had Iowa public water supplies with nitrate estimates for

70% of their person-years from 1960 onward. †

Adjusted for age group (deciles), education, and average population size of Iowa

residences. Analyses for both sexes combined are additionally adjusted for sex. ‡

Reference category.

TABLE 3. Association of the Average Nitrate Level in Iowa Drinking Water

Supplies From 1960 Onward With NHL

Average Nitrate Level

(mg/L nitrate-N)

No. of

Cases

No. of

Controls

OR†

(95% CI)

Public supplies*

0.63‡ 40 35 1.0

0.63–1.36 49 36 1.3 (0.7–2.4)

1.37–2.89 46 35 1.0 (0.5–1.9)

2.90 46 36 1.2 (0.6–2.2)

Public supplies and private wells§

0.63‡ 58 41 1.0

0.63–1.36 50 41 0.9 (0.5–1.7)

1.37–2.89 51 41 0.8 (0.4–1.4)

2.90 52 42 0.9 (0.5–1.6)

*Includes those who used Iowa public water supplies with nitrate estimates for 70% of their person-years

from 1960 onward. Assumes levels in private wells are unknown. †

Adjusted for age group (deciles), education, and sex.

‡

Reference category.

§

Includes those who used Iowa public water supplies or private wells with nitrate estimates for 70% of

their person-years from 1960 onward; nitrate levels in private wells measured at interview are assigned to all

years using the well.

Epidemiology • Volume 17, Number 4, July 2006 Non-Hodgkin Lymphoma and Drinking Water Nitrate

© 2006 Lippincott Williams & Wilkins 379
and NHL risk (Table 5). By contrast, increasing levels of

dietary nitrite intake were associated with increasing risks.

The increasing risk with nitrite intake was due almost entirely

to plant sources of nitrite (Table 5). When we evaluated the

plant nitrite association further, we found that the association

was primarily due to baked goods and cereals sources of

nitrite (highest quartile: adjusted OR 2.0; 95% CI 

1.2–3.3); there was no association for nitrite from fruits and

vegetables. The pattern in risk of NHL with dietary nitrate

and nitrite was the same for those in the diet arm who were

also included in the water nitrate analyses (not shown). In this

latter subgroup, water nitrate contributed a fairly small percent of total nitrate intake in men (median among controls 

13%; IQR 6–26) and women (median among controls 

11%, IQR 6–23).

DISCUSSION

We found no association between risk of NHL and

historical nitrate levels in Iowa public water supplies. Only a

small percentage of the study population had more than a few

years of exposure to nitrate levels at or above the maximum

contaminant level (10 mg/L), and this higher level of exposure was not associated with risk. There was no evidence of

an interaction of drinking water nitrate with lower intakes of

vitamin C or with higher intakes of red meat and processed

meats—both conditions that increase endogenous nitrosation; however, our power was limited to assess these interactions. Dietary nitrate was associated with a substantial

decreased risk of NHL. Higher nitrite intake primarily from

plant sources was associated with a substantial increased risk.

Three ecologic studies5–7 and 3 analytic epidemiologic

studies8–10 have evaluated exposure to nitrate through public

water supplies and risk of NHL. An ecologic study in Slovakia6 observed higher incidence of NHL among men and

women using public water supplies with higher average

nitrate levels (4.5–11.3 mg/L nitrate-N) a minimum of 10

years before cancer incidence. The 2 other ecologic studies

with somewhat lower nitrate levels found no consistent relationship between public supply nitrate levels and NHL

incidence.5,7 A case–control study of NHL in Nebraska8

found a positive association between the average nitrate level

in public water supplies over approximately 40 years and risk

among men and women. In the highest quartile of nitrate (4.0

mg/L nitrate-N), risk was elevated 2-fold. However, there

was little evidence of an interaction with vitamin C intake. A

case–control study of NHL in Minnesota9 with lower average

nitrate levels than the Nebraska study found an inverse

association among those drinking water supplies with the

highest level (1.5 mg/L nitrate-N). A cohort study of older

women in Iowa10 found no association between quartiles of

the average public supply nitrate over approximately 30 years

and NHL risk (highest level 2.46 mg/L nitrate-N). Interaction

with dietary factors affecting nitrosation was not evaluated.

TABLE 4. Association of the Average Nitrate Level by Vitamin C Intake With NHL*

Average Nitrate Level (mg/L)

<3 >3

No. of

Cases

No. of

Controls

OR†

(95% CI)

No. of

Cases

No. of

Controls

OR†

(95% CI)

Vitamin C; mg/d

90 31 29 1.0‡ 13 6 2.3 (0.7–7.0)

90 38 31 1.1 (0.5–2.3) 13 17 2.0 (0.6–5.9)

*Includes those who had Iowa public water supplies with nitrate estimates for 70% of their person-years from 1960 onward

and dietary information. †

Adjusted for age group (deciles), education, and sex.

‡

Reference category.

TABLE 5. Association of Dietary Intake of Nitrate and

Nitrate With NHL*

Dietary Nitrate and Nitrate

(mg/d)

No. of

Cases

No. of

Controls

OR†

(95% CI)

Nitrate

76‡ 159 98 1.0

76–113.9 116 98 0.75 (0.51–1.10)

114–169.9 111 98 0.71 (0.47–1.07)

170 80 97 0.54 (0.34–0.86)

Nitrite

0.71‡ 82 98 1.0

0.71–0.909 108 98 1.5 (1.0–2.3)

0.91–1.209 110 98 1.7 (1.1–2.7)

1.21 166 97 3.1 (1.7–5.5)

From animal sources

0.16‡ 101 98 1.0

0.16–0.259 106 98 1.0 (0.7–1.5)

0.26–0.419 123 98 1.0 (0.7–1.6)

0.42 136 97 1.0 (0.6–1.7)

From plant sources

0.48‡ 90 98 1.0

0.48–0.639 104 98 1.3 (0.8–1.9)

0.64–0.859 128 98 1.7 (1.1–2.6)

0.86 144 97 2.0 (1.2–3.5)

*Participants from all 4 centers in the diet arm of the study.

†

Adjusted for age group (deciles), education, sex, study center, race, dietary vitamin

C, and total energy. ‡

Reference category.

Ward et al Epidemiology • Volume 17, Number 4, July 2006

380 © 2006 Lippincott Williams & Wilkins
The Nebraska case–control study8 reported an inverse

association between NHL risk and dietary nitrate, which was

attenuated after adjusting for vitamin C and carotene intakes.

There was no association with dietary nitrite. The Iowa

cohort study10 observed no association with dietary nitrate

intake and did not evaluate dietary nitrite. Our findings of an

inverse association with dietary nitrate are consistent with a

reduced risk associated with antioxidants found in dark green

and other vegetables. In our study population, servings of

dark green vegetables were associated with decreased risk.13

Dark green vegetables are also important sources of dietary

folate, which was also associated with decreased risk in our

study.14 Nitrite intake from animal sources would be expected to be more strongly associated with risk if the association was due to NOC formation, because animal nitrite

sources contain higher levels of preformed NOC and NOC

precursors such as secondary amines and amino acids. However, processed meat intake was not associated with an

increased risk of NHL in our study.15 Dietary nitrite from

plant sources came largely from baked goods, cereals, and

vegetables high in nitrate. Baked good and cereal sources of

nitrite primarily accounted for the positive association between plant nitrite and NHL risk. This association may be due

to chance or to some component of breads and cereals such as

trans-unsaturated fat, which was associated with a positive

risk of NHL in a cohort study of women.20

Nitrate is a precursor in the endogenous formation of

N-nitroso compounds, most of which are animal carcinogens.21 N-nitrosoureas have been shown to induce B- and

T-cell lymphomas in animal studies.22,23 After ingestion,

nitrate is readily absorbed from the upper gastrointestinal

tract. Up to 25% is actively excreted in saliva by an active

carrier transport system, where approximately 20% is converted to nitrite by bacteria in the mouth.24 This conversion

may also occur at other sites in the body, including the distal

small intestine and the colon. Nitrosating agents can be

generated from nitrite under acidic conditions such as in the

stomach and at neutral pH through bacterial activity. In

addition, inflammatory cells produce inducible nitric oxide

synthase that can also result in the formation of nitrosating

agents and N-nitroso compounds.25 These 3 mechanisms of

endogenous nitrosation are estimated to account for roughly

40% to 75% of the total human exposure to N-nitroso

compounds.3 Other sources of human exposure include preformed N-nitroso compounds found in tobacco products,

preserved meats and fish, beer, certain occupational exposures, cosmetics, and some drugs.3

High intake of drinking water nitrate (above the maximum contaminant level) has been associated with an increased endogenous capacity to nitrosate proline.26,27 Also,

populations with high rates of esophageal, gastric cancer, and

leukemia have been found to excrete high levels of Nnitrosoproline.28–30 Elevated drinking water nitrate levels

have been associated with an increased risk of cancer of the

bladder, colon, and a few other sites; however, the studies to

date are too few to draw any conclusions.31 Nitrate intake at

the acceptable daily intake level (3.67 mg/kg body weight,

0.84 mg/kg expressed as nitrate–nitrogen) ingested largely

through drinking water results in increased urinary excretion

of N-nitroso compounds. N-nitroso compounds excretion is

increased when dietary nitrosatable precursors are ingested

simultaneously.4

In addition to contaminated drinking water, nitrate

exposure comes from the consumption of vegetables, the

primary dietary source of nitrate. The highest levels occur in

green leafy and root vegetables,32 and average daily intakes

of dietary nitrate are estimated to range from 30 to 130

mg/d.33 Although there appears to be a consistent association

between intake of high drinking water nitrate and endogenous

nitrosation capacity, intake of dietary nitrate is not likely to

increase nitrosation due to the presence of nitrosation inhibitors in vegetables.34 Antioxidants that inhibit endogenous

nitrosation include vitamin C and alpha-tocopherol. Several

epidemiologic studies reported either no association or inverse associations between dietary nitrate intake and human

cancers.8,19,35,36 Consumption of nitrate-rich vegetables, although correlated with urinary nitrate excretion, showed no

relationship with excretion of N-nitrosoproline and other

N-nitroso amino acids.29 Drinking water nitrate can constitute

the majority of nitrate intake when levels are near or above

the maximum contaminant levels.37

Strengths of our study include a lifetime water source

history, the availability of historical nitrate levels for Iowa

public water supplies, the assessment dietary sources of

nitrate and nitrite, and our ability to evaluate effect modification by vitamin C intake, meat intake, and smoking. Our

analysis of public water supply nitrate levels in the public

water supply was limited to years from 1960 onward due to

infrequent measurements earlier; however, this represented

40 years of historical data.

Our study could not evaluate risk at nitrate levels above

the maximum contaminant level due to the lack of historical

exposure data for private wells and the infrequent exposure

above this level among public water supply users. Furthermore, the large majority of the population using public water

supplies had average nitrate levels that were 5 mg/L, half

the maximum contaminant level. The group with dietary

information represented only approximately half of the study

population; therefore, small numbers of cases and controls

limited the evaluation of interaction with dietary intakes. The

finding of a modest elevated risk associated with drinking

water nitrate only among the subgroup with dietary information was unexpected and is likely due to chance.

The use of monitoring data for assessing exposure to

nitrate has several limitations. Distribution samples taken at

one or more taps quarterly or annually (as is required) may

not adequately reflect exposure if there is substantial seasonal

or spatial variation. Spatial variation across a distribution

system can occur when wells with different nitrate levels are

pumped directly into the system at different locations. Wells

are more likely to have differing nitrate levels if they come

from different sources (ie, well fields of varying depth).

However, only a small proportion of the towns included in

these analyses had multiple sources that supplied 10% of

their drinking water. Our assessment of private well nitrate

levels was limited to one measurement at the time of the

Epidemiology • Volume 17, Number 4, July 2006 Non-Hodgkin Lymphoma and Drinking Water Nitrate

© 2006 Lippincott Williams & Wilkins 381
interview, which may not reflect seasonal variations and

historical exposure levels.

In summary, levels of drinking water nitrate in public

supplies in Iowa (largely below an average level of 3 mg/L

nitrate-N) were not associated with risk of NHL overall or

among potentially susceptible subgroups. Our results for

NHL in Iowa do not confirm results from our previous study

in Nebraska in which NHL risk increased with increasing

nitrate levels in public supplies. The issue of nitrate in

drinking water and NHL risk is not resolved, but our study

offers no evidence for an increased risk for levels below 3

mg/L. Additional studies of populations with higher exposure

levels would be informative.

ACKNOWLEDGMENTS

We acknowledge the contributions of Carol Haines

(Westat, Rockville, MD) for study coordination and Shannon

Merkle and Nathan Appel (IMS, Silver Spring, MD) for

computer support. We thank David Riley and Jiji Katamneni

of the Center for Health Effects of Environmental Contaminants at the University of Iowa for providing the public utility

nitrate data.

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Ward et al Epidemiology • Volume 17, Number 4, July 2006

382 © 2006 Lippincott Williams & Wilkins