Drinking Water and Dietary Sources of Nitrate

and Nitrite and Risk of Glioma

Mary H. Ward, PhD

Ellen F. Heineman, PhD

Rodney D. McComb, MD

Dennis D. Weisenburger, MD

Objective: Dietary nitrite has been associated with increased glioma risk;

however, drinking water nitrate has not been extensively evaluated.

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

glioma in Nebraska. Water utility nitrate measurements were linked to

residential water source histories. We computed average nitrate exposure

over a 20-year period. A food frequency questionnaire was used to assess

dietary nitrate and nitrite. Results: Increasing quartiles of the average

nitrate level in drinking water were not significantly associated with risk

(adjusted odd ratios: 1.4, 1.2, 1.3). Risk was similar among those with

both higher and lower intakes of vitamin C, an inhibitor of N-nitroso

compound formation. Dietary nitrite intake was not associated with risk.

Conclusions: Our study does not support a role for drinking water and

dietary sources of nitrate and nitrite in risk of adult glioma. (J Occup

Environ Med. 2005;47:1260 –1267)

Nitrate is a precursor compound in the

formation of N-nitroso compounds

(NOC), many of which are potent

animal carcinogens.1 Of the two major

types of NOC, nitrosamines and nitrosamides, the latter, and in particular the

nitrosoureas, are strong nervous system carcinogens.1,2

NOC exposure can occur in certain

work environments through use of tobacco products and some household

products; however, the most widespread exposure to preformed NOC

comes from dietary intakes of preserved meats.3 Endogenous formation

of NOC (nitrosation) from NOC precursors has been estimated to account

for the 45–75% of exposure.3 Using a

test that measures nitrosation rates in

humans,4 two studies showed positive

associations between drinking water

nitrate ingestion and NOC excretion in

human volunteers.5,6 In another study,

those with higher nitrate levels in their

water supplies had increased blood

levels of a marker of mutagenicity.7

Drinking water can be the major

source of nitrate exposure when levels

approach the U.S. Maximum Contaminant Level (MCL) of 10 mg/L nitratenitrogen (nitrate-N).8,9 Below the

MCL, nitrate comes from dietary

sources, primarily vegetables.

Exposure to nitrate through water

consumption has not been systematically assessed in the U.S. population.

However, monitoring data from agricultural states show increases in nitrate

levels in ground water over the past 50

years caused mainly by agricultural

activity.10,11 Elevated nitrate in drinking water has been associated with an

increased risk of brain cancer in an

ecologic study12; however, another

From the Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS,

Bethesda, Maryland (Dr Ward); Division of Cancer Control and Population Sciences, National Cancer

Institute, Bethesda, Maryland (Dr Heineman); and Department of Pathology and Microbiology,

University of Nebraska, Medical Center, Omaha, Nebraska (Drs McComb, Weisenburger).

Address correspondence to: Mary H. Ward, PhD, Occupational and Environmental Epidemiology

Branch, National Cancer Institute, 6120 Executive Blvd, EPS-8104, Bethesda, MD 20892-7240;

E-mail: wardm@mail.nih.gov.

Copyright © by American College of Occupational and Environmental Medicine

DOI: 10.1097/01.jom.0000184879.67736.ae

1260 Nitrate, Nitrite, and Glioma • Ward et al
ecologic study13 and a case–control

study14 found no association with

higher nitrate levels in public water

supplies. Most epidemiologic studies

of drinking water nitrate and cancer

have been ecological or have lacked

historical information on nitrate exposure.15 The formation of NOC

is inhibited by the consumption of

antioxidants found in fruits and

vegetables,16 indicating that dietary information should be considered in addition to the assessment of drinking

water nitrate exposures.

We conducted a case–control study

of adult glioma in eastern Nebraska, a

state with elevated nitrate levels in

ground water, to further evaluate the

association of adult glioma with drinking water nitrate exposure. Dietary intakes of nitrate, nitrite, and nitrosation

inhibitors also were assessed.

Materials and Methods

We conducted a population-based

case– control study of adult glioma in

the 66 counties of eastern Nebraska.

The study population has been described previously.17 Controls were

selected randomly from controls in

a previous population-based case–

control study of hematopoietic cancers in eastern Nebraska18 and were

re-interviewed at the time of the case

interviews. Simultaneously, we also

enrolled a population-based series of

adenocarcinomas of the stomach and

esophagus.19,20

Cases were white men and women

ages 21 or older who had been newly

diagnosed with glioma (ICD-O codes

938-948) between July 1, 1988, and

June 30, 1993. Cases were limited to

white residents of eastern Nebraska

during 1983–1986 because the control subjects were selected during

that time period and other ethnic

groups had been excluded because of

expected small numbers. Cases from

1988 through 1990 were identified

from the Nebraska Cancer Registry,

whereas cases from 1991 through

June 30, 1993, were identified by

reviewing discharge diagnoses and

pathology records at 11 hospitals in

Omaha and Lincoln. From 1987–

1988, 94% of the gliomas in the

study area were treated in these hospitals. To insure that we captured all

gliomas, we requested records for

adult primary brain cancers (ICD-O

Topology codes 191-191.9) from the

Registry and participating hospitals.

From the Registry, we also requested

any records coded as gliomas

(ICD-O Morphology codes 938-

948.1), pineal tumors (codes 936),

gangliogliomas (code 950.5), neurocytomas (950.6), and neuroblastomas from other and unspecified parts

of the nervous system (ICD-O Morphology code 950.0 and Topology

code 192).

Cases were reviewed by a neuropathologist (R.D.M.) to identify eligible cases (only gliomas) and to

determine the histological subtype.21

Of the eligible interviewed cases

87% were astrocytic (56% of all

cases were glioblastoma multiforme), 8% were oligodendrogliomas, 2% ependymomas, 2% mixed

gliomas, and 1% other unspecified

gliomas. Of the 282 eligible cases,

interviews were obtained for 251

(89%; 112 women and 139 men).

Because of the severity of the disease, interviews were conducted with

the next-of-kin or proxies for 76% of

cases. The large majority of proxy

respondents were either the spouse

(62%) or other first-degree relatives (33%).

We selected a stratified random

sample from the previous populationbased control subjects as described

previously.17 We over-sampled living

controls to provide more power for

analyses by respondent type. For the

youngest cases, we had insufficient

numbers of control subjects; therefore, new controls born during the

years 1967 through 1971 were identified from death certificates (n  20,

deaths in 1988 –1993) and from random digit dialing (n  3) by the

same methods as in the earlier study.

Of the 606 eligible controls, 503

(83%) were successfully interviewed.

The response rate in the original study

was 87%, giving an overall control

response rate of 75%. Five control

subjects were excluded after the

interview because of a reported previous diagnosis of a brain tumor,

leaving 498 control subjects in the

analysis. Among these, 60% of the

interviews were conducted with

proxies. Proxy respondents were primarily the spouse (45%) or other

first-degree relatives (46%).

Telephone interviews were conducted during 1992 to 1994. Interviewers obtained information about

agricultural exposures, occupational

and residential histories, usual adult

diet, tap water intake, and other factors. For each residence, the interviewer asked about the primary

source of drinking water (public supply, private well, bottled water,

other). Water source histories were

collected from birth through the residence in 1985, the time period of the

previous study.

We used a 48-item food-frequency

questionnaire that was a modified version of the short Block Health Habits

and History Questionnaire22 with the

addition of foods high in nitrate and

nitrite. The nitrate and nitrite contents

of the food items were determined

from the literature.23–27 Those who

had missing or unknown intakes for

20% of the food items were excluded from the dietary analyses. We

adjusted for energy intake by including

total calories in the regression models

as a continuous variable. Adjustment

using the residual method28 gave similar results. The results for dietary nitrate and nitrite intakes have been

reported for the entire study population.17 Here, we present results for the

population in the drinking water analyses and we evaluate animal and plant

sources of nitrite separately. We also

estimated the contribution of drinking

water and diet sources of nitrate to

endogenous nitrite formation.

Nitrate levels in many Nebraska

public water supplies (PWS) were

measured in 1947 and periodically

thereafter as previously described.29

Nitrate levels were measured using

the phenol disulfonic acid method30

before the late 1970s and the cadmium reduction method31 thereafter.

JOEM • Volume 47, Number 12, December 2005 1261
Nitrate levels in drinking water are

expressed as mg/L nitrate-nitrogen

(nitrate-N).

We had nitrate measurements for

samples taken from the distribution

system and at the groundwater wells.

If available, we used the distribution

measurements to calculate an annual

mean nitrate level; otherwise, measurements from wells serving the

utility in that year were averaged.

For years with no nitrate measurements, we assigned an estimate

based on the annual mean nitrate

concentration for the PWS in neighboring years. We calculated a

weighted average where the weights

were 1.0, 0.75, 0.5, and 0.25 depending on whether the annual mean was

1 to 2, 3 to 4, 5 to 6, or 7 to 9 years,

respectively from the year missing

measurement data. No cases reported

using bottled water as their primary

drinking water source and use was

very limited among controls (mean

years  SD, 0.14  1.63). Nitrate

levels were assumed to be zero in

bottled water and years of bottled

water use were included in the calculation of average PWS nitrate levels. We assigned an unknown nitrate

level for communities outside of Nebraska, and for years in which the

respondent reported using a private

well.

Of the 236 Nebraska PWS included in these analyses, 73 had one

or more nitrate measurement in the

1940s, 29 had data in the 1950s, 121

in the 1960s, and 122 in the 1970s.

All but three PWS had monitoring

data in the 1980s. Among cases and

control subjects in the nitrate analyses, the total number of years with

nitrate data and the number of measurements per decade (1940s to

1980s) was similar (data not shown).

Study participants who had used a

private well in 1985 (63 cases, 72

controls) were asked to supply a tap

water sample from that well. Those

who agreed were sent a sampling kit

and instructions. A total of 47 cases

(75%) and 43 controls (60%) had

samples analyzed. The nitrate analysis

was done by the Nebraska Department

of Health Laboratories using the cadmium reduction method.31

We estimated the odds ratio (OR)

using logistic regression analysis.

Ninety-five percent confidence intervals (CI) were computed using Wald

intervals.32 In all of the analyses of

drinking water nitrate levels, the

widths of the confidence intervals

given are likely to be narrower than

they would have been had more elaborate variance adjustment procedures

been used.

Because nitrate monitoring data

were limited before the mid-1960s, we

restricted most analyses to individuals

who used Nebraska PWS after 1964.

To reduce misclassification by unknown nitrate levels, mainly because

of the use of private wells, we restricted the analyses to those with nitrate information (actual or imputed

data) for 70% or more of their personyears after 1964 (130 cases, 319 controls). Our principal measure of nitrate

exposure was an individual’s average

nitrate exposure after 1964. We also

evaluated the number of years from

1947 onward, that a PWS had nitrate

levels 5 mg/L and 10 mg/L, the

regulatory limit.

We calculated PWS nitrate exposure from 1965 through 1985, the

time frame of the previous study

resulting in a lag of 3 to 8 years

between the exposure period and

case diagnosis. To ensure a comparable exposure lag period for cases

and controls in the PWS analyses, we

excluded 100 (31%) controls who

died before the case ascertainment

period. We found no difference in

the results; therefore, we present our

findings with all 319 controls.

We computed water nitrate intake

by multiplying the usual tap water

intake (including coffee, tea, and other

beverages made with tap water) by the

average PWS nitrate level for those

with known intakes of tap water and

coffee (the major contributors to tap

water intake). An estimated 5% of

nitrate intake is endogenously converted to nitrite.3 We estimated endogenous nitrite from water nitrate intake

only because the antioxidants present

in diet nitrate sources inhibit the formation of NOC. We summed preformed diet nitrite and endogenous

nitrite from drinking water to give total

nitrite intake.

All exposure estimates were adjusted for year of birth in four categories (1915; 1915 to 1924, 1925 to

1939, 1940), education (less than

high school, high school, some college

or vocational school, college graduate/

postgraduate), and farming (ever live

or work on a farm). The dietary analyses also were adjusted for a familial

history of brain cancer, calories, carotenoids, and fiber. Dietary intake of

vitamin C and smoking were evaluated

for effect modification of the water

nitrate association. We did the major

analyses separately for self- and proxyrespondents and found no meaningful

differences by respondent type; therefore, we present results adjusted for

respondent type. We analyzed the largest histological subgroups of gliomas,

glioblastoma multiforme, and other astrocytomas separately. The numbers of

other histological subgroups were too

few to evaluate separately. The results

for glioblastoma multiforme and astrocytomas were very similar to those for

all gliomas so only the results for all

gliomas are presented.

Results

Characteristics of Persons

Included in the Drinking Water

Nitrate Analyses

A greater proportion of cases in the

PWS analyses were women compared

with the overall study population (Table 1). As expected, those in the water

analysis group had used Nebraska

public water supplies for more years

and private wells for fewer years than

the overall study population. Those in

the PWS analysis tended to have more

years of formal education; fewer cases

and control subjects had ever lived or

worked on a farm. Among men included in the PWS analysis, fewer

cases had lived or worked on a farm

compared with controls, whereas, the

opposite was true in the study population overall. The analysis group and

1262 Nitrate, Nitrite, and Glioma • Ward et al
study population overall were similar

with respect to a family history of

cancer, the type of respondent providing the interview, and dietary intakes

of nitrate, nitrite, carotenes and fiber

(not shown).

Public Water Supply

Nitrate Analyses

Average nitrate levels from 1965

to 1984 ranged from 0 to 12 mg/L.

Among control subjects, the median

was 2.58 and the interquartile range

(IQR) was 2.38 – 4.32. Increasing

quartiles of average nitrate were not

associated with risk of glioma (Table

2). Results were similar for men and

women (not shown). Water nitrate

intake (mg nitrate-N/d) calculated by

multiplying daily tap water intake by

the average PWS nitrate level ranged

from 0.2 to 39 mg/d (median 5.1;

IQR 3.1– 8.6) and was also not associated with risk (not shown).

A large number of participants

were long-term residents of Omaha

or Lincoln, the two largest cities.

Long-term residents of Omaha were

in the second quartile of average

nitrate exposure, whereas long-term

residents of Lincoln were in the fourth

quartile. To determine whether residence in these cities might confound

the nitrate association, we excluded

long-term residents of these cities

and re-evaluated the PWS nitrate association. The ORs were 1.0, 1.2,

and 0.7 for increasing quartiles of the

average nitrate level compared to the

lowest quartile.

A total of 45 (35%) cases and 102

(32%) controls in the PWS nitrate

TABLE 1

Characteristics of the Study Population Included in the Public Water Supply Nitrate Analysis and the

Total Study Population*

Population in PWS Analyses† Overall Study Population

Controls (n  319) Cases (n  130) Controls (n  498) Cases (n  251)

Men 180 (56.4) 60 (46.2) 283 (56.8) 139 (55.4)

Women 139 (43.6) 70 (53.8) 215 (43.2) 112 (44.6)

Year of birth

Before 1915 104 (32.6) 13 (10.0) 158 (31.7) 32 (12.7)

1915–1924 80 (25.1) 38 (29.2) 121 (24.3) 73 (29.1)

1925–1939 78 (24.4) 44 (33.8) 116 (23.3) 74 (29.5)

1940 or later 57 (17.9) 35 (27.0) 103 (20.7) 72 (28.7)

Nebraska public water

Median yr (IQR) 41 (28 –54) 42 (31–53) 30 (14 – 46) 28 (7– 44)

Nebraska private wells

Median yr (IQR) 0 (0 –20) 0 (0 –12) 8 (0 –32) 0 (0 –30)

Unknown source

Median yr (IQR) 5 (0 –7) 0 (0 – 4.5) 6 (1–7) 0 (0 –5)

Education

12 yr 84 (26.4) 15 (11.5) 151 (30.3) 44 (17.5)

High school 101 (31.8) 46 (35.4) 140 (28.1) 84 (33.5)

Some college, vocational technical school 81 (25.5) 40 (30.8) 116 (23.3) 68 (27.1)

College 4 yr/Postgraduate 52 (16.3) 29 (22.3) 87 (17.5) 55 (21.9)

Ever live/work on farm?

Men

Yes 101 (56.1) 28 (46.7) 179 (63.2) 93 (67.4)

No 79 (43.9) 32 (53.3) 104 (36.8) 45 (32.6)

Women

Yes 85 (61.2) 31 (44.3) 143 (66.5) 60 (53.6)

No 54 (38.8) 39 (55.7) 72 (33.5) 52 (46.4)

First-degree relative with cancer

None 137 (42.9) 58 (44.6) 230 (46.2) 103 (41.0)

Central nervous system cancer 7 (2.2) 6 (4.6) 12 (2.4) 10 (4.0)

Other cancer 146 (45.8) 57 (43.9) 214 (43.0) 119 (47.4)

Don’t know 29 (9.1) 9 (6.9) 42 (8.4) 19 (7.5)

Respondent

Self 125 (39.2) 29 (22.3) 198 (39.8) 61 (24.3)

Spouse 89 (27.9) 62 (47.7) 135 (27.1) 118 (47.0)

Daughter/son 65 (20.4) 30 (23.1) 104 (20.9) 50 (19.9)

Parent/sibling 22 (6.9) 6 (4.6) 33 (6.6) 12 (4.8)

Other relative 15 (4.7) 1 (0.8) 22 (4.4) 7 (2.8)

Other 3 (0.9) 2 (1.5) 6 (1.2) 3 (1.2)

*Percentages may not total 100 as the result of missing information.

†Population with 70% of person-yr 1965 to 1984 with public supply nitrate levels.

JOEM • Volume 47, Number 12, December 2005 1263
analysis had 1 or more years of

exposure to public supplies with a

nitrate level 10 mg/L nitrate-N

from 1947–1984 (Table 3). Compared with no exposure, there was no

association between the years of exposure to drinking water nitrate at

these levels and risk of glioma. Increasing years using a PWS with

nitrate levels of 5 mg/L was associated with nonsignificant increasing

risk up to 9 years, however there was

no association among those exposed

for 10 or more years.

We evaluated the risk of glioma

with combined exposure to quartiles of

the PWS average nitrate and usual

vitamin C intake (median, median;

Table 4). There was no evidence of an

interaction between drinking water nitrate and dietary vitamin C intake. ORs

were nonsignificantly elevated in all

categories of water nitrate and vitamin

C intake compared to those with low

water nitrate and high vitamin C intake. We obtained similar results when

we evaluated years of exposure 5

mg/L nitrate-N. We also found no

evidence of interaction by smoking

status (current/past smokers and nonsmokers—not shown).

Nitrite Intake From Diet and

Drinking Water Sources

A total of 121 cases and 285

control subjects in the PWS nitrate

analysis had adequate dietary information (20% unknown food

items). As was previously reported

for the entire study population,17

there was no association between

dietary nitrate (not shown), dietary

nitrite, and glioma risk among those in

the PWS analysis (Table 5). Higher

intake of plant nitrite was associated

with a significantly increased risk;

there was no association with animal

sources. Total nitrite as the sum of

dietary nitrite and endogenous nitrite

from drinking water nitrate was not

significantly associated with risk (Table 5). There was no evidence of an

interaction between vitamin C intake

and dietary nitrite or nitrite intake from

diet and drinking water (not shown).

Private Well Nitrate Analyses

The nitrate levels measured in private wells ranged from below the

detection limit (0.5 mg/L nitrate-N)

to 67 mg/L nitrate-N (median among

TABLE 3

Odds Ratios (OR) and 95% Confidence Intervals (CI) for Glioma and Year of

Using a Nebraska Public Water Supply With 5 and 10 mg/L Nitrate,

1947 to 1984*

Years Cases Controls OR† (95% CI)

10 mg/L

0 85 217 1.0

1– 8 25 54 1.1 (0.6 –2.1)

9 20 48 1.1 (0.6 –2.0)

5 mg/L

0 22 82 1.0

1– 4 62 127 1.3 (0.7–2.5)

5–9 20 33 1.8 (0.8 – 4.1)

10 26 77 1.1 (0.5–2.2)

*Includes those with 70% or more of their person-yr after 1964 with Nebraska public water

supply nitrate estimates.

†Odds ratios adjusted for year of birth, gender, respondent type, education, and ever

live/work on a farm.

TABLE 4

Odds Ratios (OR) and 95% Confidence Intervals (CI) for Glioma Associated With The Combined Average Nitrate Level In

Nebraska Public Water Supplies (1965 to 1984) and Dietary Vitamin C Intake*

Vitamin C mg/d

Public Water Supply Nitrate Level (mg nitrate-N/L)

<2.36 Cases/Controls

OR (CI)

2.38 –2.57 Cases/Controls

OR† (CI)

2.58 – 4.32 Cases/Controls

OR (CI)

>4.32 Cases/Controls

OR (CI)

113 8/38

1.0

22/41

2.4 (0.8 – 6.9)

13/33

2.0 (0.7– 6.0)

13/31

2.1 (0.7– 6.4)

113 15/33

2.3 (0.8 – 6.9)

16/34

1.6 (0.5– 4.8)

17/38

1.7 (0.6 – 4.7)

17/37

2.0 (0.7–5.8)

*Includes those with 70% or more of their person-yr after 1964 with Nebraska public water supply nitrate estimates.

†ORs adjusted for year of birth, gender, respondent type, education, and ever live/work on a farm.

TABLE 2

Odds Ratios and 95% Confidence Intervals for Glioma, and the Average Nitrate

Level in Nebraska Public Water Supplies, 1965 to 1984*

mg nitrate-N/L Cases Controls OR† (95% CI)

2.38 24 79 1.0

2.38 –2.57 39 79 1.4 (0.7–2.7)

2.58 – 4.32 33 81 1.2 (0.6 –2.3)

4.32 34 80 1.3 (0.7–2.6)

*Includes those with 70% or more of their person-yr after 1964 with Nebraska public water

supply nitrate estimates.

†ORs adjusted for age, gender, respondent type, education, and ever live/work on a farm.

1264 Nitrate, Nitrite, and Glioma • Ward et al
controls 2.3; IQR 0.2–7.4; 90th percentile 16.6). There was no association between increasing tertiles of

the nitrate level and glioma risk (not

shown). We compared those with

private well nitrate levels at or above

10 mg/L (16% of controls; 15% of

cases) to those whose wells were

lower but found no association with

risk (OR  1.2, 95% CI  0.4 – 4.1).

The small number of individuals

prohibited the evaluation of effect

modification.

We evaluated the duration of private well use in Nebraska and risk of

glioma because private well users

often have higher water nitrate exposure than those using public supplies.

Results differed for men and women.

Among men, there was a significantly 2.5-fold increase among those

with 30 or more years using private

wells for their drinking water (Table

6). Among women, duration of private well use was inversely associated with risk. We evaluated years of

well use after 1964 (Table 6) because

nitrate levels in many water supplies

increased between the 1960s and

1980s.29,33 Fifteen or more years of

well use after 1964 was associated

with a significant 3.1-fold increased

risk among men but there was no

association among women. Shallow

wells (100 feet) tend to have higher

nitrate levels than deeper wells.34 Only

17 (6.8%) cases and 98 (19.7%) control subjects had ever used a shallow

well for their drinking water. We observed a pattern of risk similar to total

years of well use; among men the OR

for 30 or more years was elevated

(OR  1.7, 95% CI  0.6 – 4.7) and

among women the OR was inverse

(OR  0.4, 95% CI  0.1–1.4).

TABLE 5

Odds Ratio (OR) and 95% Confidence Intervals (CI) for Glioma and

Intakes of Nitrite

mg/d Cases/Controls OR (CI)*

Preformed diet nitrite†

0.70 38/67 1.0

0.70 to 0.94 27/74 0.8 (0.4 –1.7)

0.94 –1.19 23/71 1.0 (0.4 –2.3)

1.19 33/73 1.2 (0.5–3.2)

Diet nitrite from animal sources†

0.29 29/68 1.0

0.29 to 0.46 25/72 0.8 (0.4 –1.6)

0.46 – 0.63 31/70 1.3 (0.6 –2.7)

0.63 36/75 1.3 (0.6 –2.9)

Diet nitrite from plant sources†

0.31 34/68 1.0

0.31 to 0.43 29/74 1.0 (0.5–2.1)

0.43– 0.59 28/68 3.2 (1.2– 8.3)

0.59 30/75 2.8 (1.0 – 8.2)

Preformed diet intake plus endogenous

nitrite from water nitrate‡

1.4 21/70 1.0

1.4 to 2.1 43/71 1.8 (0.9 –3.6)

2.1 to 3.3 28/71 1.1 (0.5–2.3)

3.3 28/71 1.1 (0.5–2.5)

*Odds ratios adjusted for year of birth, gender, respondent type, ever live/work on a farm,

education, beta-carotene, fiber, calories.

†Includes those with 70% or more of their person-yr after 1964 with Nebraska public water supply

nitrate estimates and adequate dietary information (excludes those with 20% unknown foods).

‡Includes those with 70% or more of their person-yr after 1964 with Nebraska public water

supply nitrate estimates and adequate dietary and tap water information (excludes those with

20% unknown foods and unknown water/coffee intake).

TABLE 6

Odds Ratios (ORs) and 95% Confidence Interval (CI) for Glioma With Duration of Private Well Use in Nebraska

All Men Women

Cases Controls OR (95% CI)* Cases Controls OR (95% CI)† Cases Controls OR (95% CI)†

Years of private well

use in Nebraska‡

0 105 178 1.0 48 110 1.0 57 68 1.0

1–9 12 26 0.6 (0.3–1.4) 8 16 1.1 (0.4 –3.0) 4 10 0.2 (0.05–1.0)

10 –19 27 52 0.8 (0.4 –1.5) 10 24 0.9 (0.4 –2.4) 17 28 0.5 (0.2–1.3)

20 –29 18 56 0.6 (0.3–1.1) 13 30 1.0 (0.4 –2.4) 5 26 0.2 (0.04 – 0.7)

30 64 134 1.4 (0.8 –2.5) 42 77 2.5 (1.2–5.4) 22 57 0.5 (0.2–1.3)

Years of private well

use in Nebraska

after 1964‡

0 168 363 1.0 83 206 1.0 85 157 1.0

1–14 28 61 1.2 (0.7–2.0) 16 35 1.2 (0.6 –2.6) 12 26 1.1 (0.4 –2.5)

15 55 74 2.3 (1.4 –3.6) 40 42 3.1 (1.7–5.7) 15 32 1.3 (0.6 –2.8)

*ORs adjusted for year of birth, gender, respondent type, ever live/work on a farm, and education.

†ORs adjusted for year of birth, respondent type, ever live/work on a farm, and education.

‡Excludes those with private well use outside of Nebraska.

JOEM • Volume 47, Number 12, December 2005 1265
Discussion

The average nitrate level in public

drinking water supplies in Nebraska

during a 20-year period was not associated with glioma risk. Likewise,

years of exposure to nitrate at or

above the MCL of 10 mg/L nitrate-N

and half the MCL was not significantly associated with risk; the duration of exposure above the MCL

generally was low.

Elevated nitrate in drinking water

was associated with an increased risk

of brain cancer in a small area ecologic study in Yorkshire, England12;

however, another ecologic study in

Canada13 found no association. The

highest average exposure quartile in

the Yorkshire study (5.3 mg/L nitrate-N) was slightly higher than our

highest quartile level. A case-control

study in Germany14 found no association between glioma risk and average nitrate levels in PWS that were

similar to average levels in our study.

In our study, the nitrate level in

private wells at the time of the interview was not associated with risk of

glioma; however, a current well measurement may not reflect long-term

exposure. Longer duration of use of

Nebraska private wells, particularly

after 1964, was associated with an

elevated risk of glioma among men

but not women. In our study population, the duration of private well use

was strongly correlated with the duration of farming and it was not

possible to adjust for farming duration. Occupational surveys indicate

that farmers have a higher risk of

brain cancer compared with the general population.35 Residual confounding by factors associated with

long-term farming may explain the

increased risk we observed.

Our study does not support dietary

nitrite intake as a risk factor for adult

glioma risk. Plant sources of nitrite,

which consist mainly of baked goods

and cereals, would not be expected to

preferentially result in increased

NOC formation compared with animal sources. Therefore, the significant association for plant nitrite was

unexpected. Further, nitrite intake

which incorporated the contribution

of drinking water nitrate to endogenous nitrite formation was also not

associated with risk. We did not observe any evidence of an interaction

between nitrite intake and vitamin C.

Other case– control studies that estimated dietary nitrite have found

mixed results.36 – 41 A study by Lee

et al40 found a significantly elevated

risk among men with high nitrite

intake and low vitamin C intake. A

recent meta-analysis of nine studies

evaluating cured meat found a

pooled RR of 1.48 (95% CI  1.20 –

1.83) for glioma among adults ingesting high levels of cured meat.42

However, the authors concluded that

the failure of most studies to adjust

for total energy intake might have

led to spurious positive associations

in some studies.

Strengths of our analysis include the

high response rates among cases and

controls, a lifetime water source history, a historical nitrate database for

public water supplies, our evaluation

of effect modification by vitamin C

intake and smoking, and our ability to

estimate nitrite intake from diet and

drinking water. Our analysis of community supply nitrate levels was limited to considering exposures that

occurred over the course of approximately 20 years due to infrequent measurements before 1965. Our study had

little power to evaluate risk at nitrate

levels above the MCL because of the

few study participants who were using

a private well at the time of the interview and because of the infrequent

exposure above this level among public water supply users.

Because of the high mortality rate

among glioma cases, we conducted

the majority of interviews with proxies, most of whom were spouses

or other first-degree relatives. We

found no substantial differences in

our results by respondent type. We

had both living and deceased control

subjects in our study. Controls chosen from mortality files have been

shown to have a poorer diet and a

higher proportion of heavy drinkers

and smokers compared to living persons of the same age43 which would

result in underestimation of the risk

for these factors. However, these differences between living and deceased controls would be unlikely to

affect our drinking water nitrate

analyses. The dates-of-death of some

deceased controls preceded the case

ascertainment period by 3 to 5 years.

Because of the longer time between

the death date and interview date

for some deceased controls compared with deceased cases, proxies

for these controls may have had

more difficulty in recalling information. However, excluding controls

whose date-of-death preceded the

case ascertainment period did not

materially change our results.

Nitrosamines must be metabolized

before they are biologically active,

whereas nitrosamides are chemically

unstable, acting at the site of formation.44 Nitrosamides are particularly

strong neurocarcinogens in animal

studies when administered in utero.2

However, the animal and epidemiologic evidence for the carcinogenicity

of NOCs for adult glioma is less clear.

In summary, we found no association between drinking water nitrate

and glioma risk. Our study had limited

power to evaluate PWS nitrate levels

greater than approximately 5 mg/L

nitrate-N. Dietary nitrite intake was

not associated with risk and incorporating the contribution of water and

dietary nitrate to endogenous nitrite

formation did not change our findings.

Large studies in populations with

higher exposures to drinking water nitrate and dietary nitrite are warranted

to further evaluate the NOC hypothesis

in relation to adult glioma.

Acknowledgments

The authors thanks Robert Saal, Casey

Boudreau, and Carol Russell for assistance in

study management and coordination; Monica

Seeland and other staff of the Nebraska Cancer Registry for providing data; John Blosser

and the staff at the Nebraska Department of

Health laboratory; study interviewers and

support staff for their diligent work; and the

many physicians and study participants who

cooperated in this study.

1266 Nitrate, Nitrite, and Glioma • Ward et al
This research was supported by the Intramural Research Program of the NIH, National

Cancer Institute.

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