# 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 ) 

# N itrate is a precursor compound in the formation of N-nitroso compounds (NOC), many of which are potent animal carcinogens. 1 Of the two majortypes of NOC, nitrosamines and nitrosamides, the latter, and in particular thenitrosoureas, are strong nervous system carcinogens. 1,2

NOC exposure can occur in certainwork environments through use of tobacco products and some householdproducts; however, the most widespread exposure to preformed NOCcomes from dietary intakes of preserved meats. 3 Endogenous formationof 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 levelsapproach 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 waterconsumption 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 study 12 ; 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 alecologic study 13 and a case–control study 14 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 lackedhistorical 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 in

formation 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 theassociation 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 Nebraska 18 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 Nebraskaduring 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 thestudy 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 partsof 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 allcases 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 randomsample 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 insufficientnumbers of control subjects; therefore, new controls born during theyears 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 theinterview 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 withproxies. 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 adultdiet, tap water intake, and other factors. For each residence, the interviewer asked about the primarysource of drinking water (public supply, private well, bottled water, other). Water source histories werecollected from birth through the residence in 1985, the time period of the previous study. We used a 48-item food-frequencyquestionnaire 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 sim

ilar results. The results for dietary nitrate and nitrite intakes have beenreported for the entire study population. 17 Here, we present results for thepopulation 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 1261Nitrate 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 annualmean 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 nitrateconcentration for the PWS in neighboring years. We calculated a weighted average where the weightswere 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 bottledwater use were included in the calculation of average PWS nitrate levels. We assigned an unknown nitratelevel 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 andcontrol subjects in the nitrate analyses, the total number of years withnitrate 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 thanthey 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 becauseof the use of private wells, we restricted the analyses to those with nitrate information (actual or imputeddata) 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 andcase 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% ofnitrate intake is endogenously converted to nitrite. 3 We estimated endog

enous 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 liveor work on a farm). The dietary analyses also were adjusted for a familialhistory 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 majoranalyses separately for self- and proxyrespondents and found no meaningfuldifferences by respondent type; therefore, we present results adjusted forrespondent 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 resultsfor 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 comparedwith 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 orworked on a farm. Among men included in the PWS analysis, fewer cases had lived or worked on a farm compared with controls, whereas, theopposite was true in the study population overall. The analysis group and 1262 Nitrate, Nitrite, and Glioma • Ward et alstudy population overall were similar with respect to a family history ofcancer, 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 fourthquartile. To determine whether residence in these cities might confound the nitrate association, we excluded long-term residents of these citiesand 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 1263analysis had 1 or more years of exposure to public supplies with a nitrate level  10 mg/L nitrate-Nfrom 1947–1984 (Table 3). Compared with no exposure, there was noassociation between the years of exposure to drinking water nitrate atthese 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 aninteraction 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 lowwater 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 smokingstatus (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 nitrateanalysis 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 notsignificantly 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 alcontrols 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 usersoften 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 (Table6). 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 Only17 (6.8%) cases and 98 (19.7%) control subjects had ever used a shallowwell 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 Nebraskaduring 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-Nand 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 riskof brain cancer in a small area ecologic study in Yorkshire, England 12 ; however, another ecologic study in Canada 13 found no association. The highest average exposure quartile inthe Yorkshire study (5.3 mg/L nitrate-N) was slightly higher than our highest quartile level. A case-control study in Germany 14 found no associ

ation between glioma risk and average nitrate levels in PWS that were similar to average levels in our study.

In our study, the nitrate level inprivate wells at the time of the interview was not associated with risk ofglioma; 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 menbut not women. In our study population, the duration of private well usewas strongly correlated with the duration of farming and it was notpossible to adjust for farming duration. Occupational surveys indicate that farmers have a higher risk ofbrain cancer compared with the general population. 35 Residual con

founding 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 increasedNOC formation compared with animal sources. Therefore, the significant association for plant nitrite was unexpected. Further, nitrite intake which incorporated the contributionof drinking water nitrate to endogenous nitrite formation was also notassociated 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 andcontrols, 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 anddrinking water. Our analysis of community supply nitrate levels was limited to considering exposures thatoccurred 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 usinga private well at the time of the interview and because of the infrequentexposure above this level among public water supply users.

Because of the high mortality rate among glioma cases, we conductedthe 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 controlsubjects 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 riskfor 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 datefor some deceased controls compared with deceased cases, proxies for these controls may have hadmore 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 chemicallyunstable, acting at the site of formation. 44 Nitrosamides are particularly strong neurocarcinogens in animal studies when administered in utero. 2However, 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 wasnot 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 withhigher 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 alThis research was supported by the Intramural Research Program of the NIH, National
Cancer Institute. 

### References

1. Bogovski P, Bogovski S. Animal species

in which N-nitroso compounds induce

cancer. Int J Cancer. 1981;27:471– 474.2. Tomatis L. Prenatal carcinogenesis. In: Kakundaga T, Sugimura T, Tomatis L, Yamasaki H, eds. Cell Diff Genes and Cancer. Sci Pub No 92. Lyon: IARC; 1988:121–132. 3. Tricker AR. N-Nitroso compounds and man: sources of exposure, endogenous formation and occurrence in body fluids.
Eur J Cancer Prev . 1997;6:226 –268.4. Ohshima H, Bartsch H. Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitroso-proline excreted in the
urine. Cancer Res. 1981;41:3658–3662. 5. Mirvish SS, Grandjean AC, Moller H, et
al. N -nitrosoproline excretion by ruralNebraskans drinking water of varied nitrate content. Cancer Epidemiol Biomarkers Prev . 1992;1:455– 461. 6. Moller H, Landt J, Perdersen E, et al.Endogenous nitrosation in relation to nitrate exposure from drinking water and diet in a Danish rural population. Cancer
Res . 1989;49:3117–3121. 7. Van Maanen JMS, Welle IJ, Hageman G, et al. Nitrate contamination of drinking water: Relationship with HPRT variantfrequency in lymphocyte DNA and urinary excretion of N -nitrosamines. Environ Health Perspect. 1996;104:522–528. 8. Chilvers C, Inskip H, Caygill C et al. A survey of dietary nitrate in well-water

users. Int J Epidemiol. 1984;13:324 –31. 9. Moller H, Landt J, Jensen P, et al. Nitrate exposure from drinking water and diet in a Danish rural population. Int J Epidemiol . 1989;18:206 –211. 10. Hallberg GR, Riley DR, Kantamneni JR,
et al. Assessment of Iowa Safe Drinking Water Act monitoring data: 1988 –1995.The University of Iowa Hygienic Laboratory Research Report No. 1996;97–1. 11. Nolan BT, Ruddy BC, Hitt KJ, et al. Risk of nitrate in groundwaters of the United States—a national perspective. Environ Sci Technol . 1997;31:2229 –2236. 12. Barrett JH, Parslow RC, McKinney PA, et al. Nitrate in drinking water and the incidence of gastric, esophageal, and brain cancer in Yorkshire, England. Cancer Causes Control. 1998;9:153–159.13. Van Leeuwen JA, Waltner-Toews D, Abernathy T, et al. Associations between stomach cancer incidence and drinking water
contamination with atrazine and nitrate in Ontario (Canada) agroecosystems, 1987–
1991. Int J Epidemiol. 1999;28:836–840. 14. Steindorf K, Schlehofer B, Becher H, et al. Nitrate in drinking water. A case-control study on primary brain tumours with anembedded drinking water survey in Germany. Int J Epidemiol. 1994;23:451–457. 15. Cantor KP. Drinking water and cancer.
Cancer Causes Control. 1997;8:292–308.16. Bartsch H, Frank N. Blocking the endogenous formation of N-nitroso compounds and related carcinogens. In: Stewart BW, McGregor D, Kleihues P, eds. Principles of Chemoprevention . IARC Scientific Publications No. 139. Lyon: International Agency for Research on Cancer; 1996. 17. Chen H, Ward MH, Tucker KL, et al. Diet and risk of adult glioma in eastern Nebraska, United States. Cancer Causes
Control . 2002;13:647– 655. 18. Zahm SH, Weisenburger DD, Babbitt PA,et al. A case-control study of nonHodgkin’s lymphoma and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in
eastern Nebraska. Epidemiology. 1990;1:
349 –356. 19. Ward MH, Sinha R, Heineman EF, et al.
Risk of adenocarcinoma of the stomach and esophagus with meat cooking method and doneness preference. Int J Cancer

. 1997;71:14 –19. 20. Chen H, Ward MH, Graubard BI, et al. Dietary patterns and adenocarcinoma of the stomach and esophagus. Am J Clin
Nutr. 2002;75:137–144. 21. Kleihues P, Burger PC, Scheithauer BW.
The new WHO classification of brain

tumours. Brain Pathol . 1993;3:255–268. 22. Block G, Hartman AM, Naughton D. A reduced dietary questionnaire: development
and validation. Epidemiology. 1990;1:58–64. 23. National Academy of Sciences. The Health Effects of Nitrate, Nitrite and N-Nitroso Compounds. Washington, DC: National Academy Press; 1981. 24. Howe GR, Harrison L, Jain M. A short diet history for assessing dietary exposure to N-nitrosamines in epidemiologic studies. Am J Epidemiol. 1986;124:595– 602. 25. Panalaks T, Iyengar JR, Sen NP. Nitrate, nitrite, and dimethylnitrosamine in cured meat products. J Assoc Off Anal Chem

. 1973;56:621– 625. 26. Panalaks T, Iyengar JR, Donaldson BA,et al. Further survey of cured meat products for volatile N-nitrosamines. J Assoc Off Anal Chem . 1974;57:806 – 812. 27. White JW Jr. Relative significance of dietary sources of nitrate and nitrite. J Agric Food Chem . 1975;23:886 – 891.28. Willett WC, Howe GR, Kushi LH. Adjustment for total energy intake in epidemiologic
studies. Am J Clin Nutr. 1997;65:1220S– 1228S; discussion 1229S–1231S. 29. Ward MH, Mark SD, Cantor KP, et al. Drinking water nitrate and the risk of non-Hodgkin’s lymphoma. Epidemiology . 1996;7:465– 471.
30. American Public Health Association. Standard Methods for the Examination of Water
and Wastewater. 13th ed

. New York: American Public Health Association; 1971.
31. American Public Health Association. Standard Methods for the Examination of Water
and Wastewater. 14th ed. New York: American Public Health Association; 1976. 32. Armitage P, Berry G. Statistical Methods
in Medical Research , Oxford: Blackwell Scientific Publications; 1987. 33. Exner ME, Spalding R. Occurrence of Pesticides and Nitrate in Nebraska’s Groundwater. Institute of Agriculture and Natural Resources, University of Nebraska; 1990. 34. Kross BC, Hallberg GR, Bruner DR, et al. The nitrate contamination of private
well water in Iowa. Am J Public Health

. 1993;83:270 –272. 35. Zahm SH, Ward MH, Blair A. Pesticides
and cancer. Occup Med. 1997;12:269 –289. 36. Giles GG, McNeil JJ, Donnan GA, et al. Dietary factors and the risk of glioma in adults: results of a case-control study in Melbourne, Australia. Int J Cancer

. 1994;59:357–362. 37. Kaplan S, Novikov I, Modan B. Nutritionalfactors in the etiology of brain tumors: potential role for nitrosamines, fat, and cholesterol. Am J Epidemiol. 1997;146:832–841. 38. Blowers L, Preston-Martin S, Mack W. Dietary and other lifestyle factors ofwomen with brain gliomas in Los Angeles County (California, USA). Cancer Causes Control. 1997;8:5–12. 39. Preston-Martin S, Pogoda JM, Mueller BA, et al. Maternal consumption of curedmeats and vitamins in relation to pediatric brain tumors. Cancer Epidemiol Biomarkers Prev . 1996;5:599 – 605. 40. Lee M, Wrensch M, Mike R. Dietary andtobacco risk factors for adult onset glioma in the San Francisco Bay Area (California, USA). Cancer Causes Control . 1997;8:13–24. 41. Boeing H, Schlehofer B, Blettner M, et al. Dietary carcinogens and the risk of glioma and meningioma in Germany. Int
J Cancer. 1993;53:561–565. 42. Huncharek M, Kupelnick B, Wheeler LJ. Dietary cured meat and the risk of adultglioma: a meta-analysis of nine observational studies. Environ Pathol Toxicol
Oncol . 2003;22:129 –137. 43. McLaughlin JK, Blot WJ, Mehl ES, et al.
Problems in the use of dead controls in
case-control studies I. General results. Am J Epidemiol. 1985;121:131–139.
44. Mirvish SS. Formation of N-nitroso compounds: chemistry, kinetics, and in vivo

occurrence. Tox Applied Pharmacol

. 1975;31:325–351. JOEM • Volume 47, Number 12, December 2005 1267