## O RIGINAL 

## 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 watersource 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 orred 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) 

# N itrate has steadily accumulated in our water resources largely 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 includesmany 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 thatinhibit endogenous nitrosation include vitamin C and alphatocopherol. Three ecologic studies 5–7 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 study 8 observed an elevatedrisk with nitrate levels in public water supplies; no association was observed in the other studies. Two studies 8,10 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 

### 375metropolitan area; Los Angeles County, California; and theSeattle, 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 digitdialing; 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 AnalysisThe 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 linebetween 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 main

tains 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 wasuncommon 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). Amongthese 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 andcontrols, 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 watersource. 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

© 2006 Lippincott Williams & Wilkins 

### 376

### 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. Eachstudy 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 dietaryquestionnaire. Two other cases and 2 other controls inadvertently received and returned the dietary questionnaire for atotal 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 addi

tional 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 offoods were determined from the literature, as described previously. 18,19 Daily intakes of nitrate and nitrite were calcu

lated 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 adjustedfor 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 forfewer 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 

### 377TABLE 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

© 2006 Lippincott Williams & Wilkins 

### 378substantially 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 

### 379and 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 maximumcontaminant 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 processedmeats—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 studies 5–7 and 3 analytic epidemiologic studies 8–10 have evaluated exposure to nitrate through publicwater 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 studieswith 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 Nebraska 8 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 Iowa 10 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

© 2006 Lippincott Williams & Wilkins 

### 380The Nebraska case–control study 8 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 study 10 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 ex

pected 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 NOCprecursors 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 ofnitrite 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 ofN-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 activecarrier 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 pre

formed N-nitroso compounds found in tobacco products,preserved meats and fish, beer, certain occupational exposures, cosmetics, and some drugs. 3High 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, andleukemia 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 toincrease nitrosation due to the presence of nitrosation inhibitors in vegetables. 34 Antioxidants that inhibit endogenous nitrosation include vitamin C and alpha-tocopherol. Severalepidemiologic studies reported either no association or inverse associations between dietary nitrate intake and human

cancers. 8,19,35,36 Consumption of nitrate-rich vegetables, al

though 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 ofnitrate 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 exposureabove 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 drinkingwater 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 

### 381interview, 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 Katamneniof the Center for Health Effects of Environmental Contaminants at the University of Iowa for providing the public utility nitrate data.

REFERENCES

1. Fields S. Global nitrogen: cycling out of control. Environ Health

Perspect. 2004;112:A557–A563. 2. Environmental Protection Agency. List of Contaminants and Their

MCLs. Available at: http://www.epa.gov/safewater/mcl.html. Accessed December 5, 2005.3. 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. Vermeer I, Pachen DM, Dallinga JW, et al. Volatile N-nitrosamine formation after intake of nitrate at the ADI level in combination with an amine-rich diet. Environ Health Perspect. 1998;106:459–463. 5. Law G, Parslow R, McKinney P, et al. Non-Hodgkin lymphoma and

nitrate in drinking water: a study in Yorkshire, United Kingdom. J Epidemiol Community Health . 1999;53:383–384. 6. Gulis G, Czompolyova M, Cerhan JR. An ecologic study of nitrate in

municipal drinking water and cancer incidence in Trnava District, Slovakia. Environ Res. 88:182–187. 7. Cocco P, Broccia G, Aru G, et al. Nitrate in community water supplies

and incidence of non-Hodgkin’s lymphoma in Sardinia, Italy. J Epidemiol Community Health . 2003;57:510–511. 8. Ward MH, Mark SD, Cantor KP, et al. Drinking water nitrate and the

risk of non-Hodgkin lymphoma. Epidemiology . 1996;7:465–471. 9. Freedman DM, Cantor KP, Ward MH, et al. A case–control study of

nitrate in drinking water and non-Hodgkin lymphoma in Minnesota. Arch Environ Health . 2000;55:326–329. 10. Weyer PJ, Cerhan JR, Kross BC, et al. Municipal drinking water nitrate level and cancer risk in older women: the Iowa Women’s Health Study. Epidemiology . 2001;12:327–338.11. Chatterjee N, Hartge P, Cerhan JR, et al. Risk of non-Hodgkin lymphoma and family history of lymphatic, hematologic, and other cancers. Cancer Epidemiol Biomarkers Prev . 2004;13:1415–1421. 12. Center for Health Effects of Environmental Contaminants (CHEEC). Historical Community Water Supply and Treatment Data for the State of Iowa , 3rd ed. Iowa City: The University of Iowa; 1998. 13. Keleman LE, Cerhan JR, Lim U, et al. Association of vegetables, fruitsand antioxidants with non-Hodgkin lymphoma: an NCI-SEER population-based case–control study. Am J Clin Nutr. In press.14. Lim U, Schenk M, Kelemen LE, et al. Dietary determinants of onecarbon metabolism and the risk of non-Hodgkin lymphoma: NCI-SEER Case–Control Study (1998–2000). Am J Epidemiol. 2005;162:953–964. 15. Cross AJ, Ward MH, Schenk M, et al. Meat and Meat-Mutagen Intake and Risk of Non-Hodgkin Lymphoma: Results from a NCI-SEER Case–Control Study. Carcinogenesis . 2006;27:293–297. 16. Block G, Hartman AM, Dresser CM, et al. A data-based approach to diet questionnaire design and testing. Am J Epidemiol. 1986;124:453–469. 17. Block G, Coyle LM, Hartman AM, et al. Revision of dietary analysis software for the Health Habits and History Questionnaire. Am J Epidemiol. 1994;139:1190–1196. 18. Ward MH, Zahm SH, Weisenburger DD, et al. Dietary factors and non-Hodgkin’s lymphoma in Nebraska (United States). Cancer Causes Control. 1994;5:422–432. 19. Ward MH, Cantor KP, Riley D, et al. Nitrate in public water supplies and risk of bladder cancer. Epidemiology . 2003;14:183–190. 20. Zhang S, Hunter DS, Rosner BA, et al. Dietary fat and protein in relation to risk of non-Hodgkin’s lymphoma among women. J Natl Cancer Inst

. 1999;91:1751–1758. 21. Bogovski P, Bogovski S. Animal species in which N-nitroso compounds induce cancer. Int J Cancer. 1981;27:471–474. 22. Mirvish SS, Weisenburger DD, Salmasi S, et al. Carcinogenicity of1-(2-hydroxyethyl)-1-nitrosourea and 3-Nitroso-2-oxazolidinone administered in drinking water to male MRC-Wistar rats: induction of bone, hematopoietic, intestinal, and liver tumors. J Natl Cancer Inst. 1987;78: 387–393. 23. Mirvish SS, Weisenburger DD, Joshi SS, et al. 2-hydroxyethylnitrosourea induction of B-cell lymphoma in female Swiss mice. Cancer Lett. 1990;54: 101–106. 24. Spiegelhalter B, Eisenbrand G, Preussman R. Influence of dietary nitrateon nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet Toxicol. 1976;14:545–548. 25. Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res . 1994;305:253–264. 26. 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. 27. Mirvish SS, Grandjean AC, Moller H, et al. N-Nitrosoproline excretion by rural Nebraskans drinking water of varied nitrate content. Cancer Epidemiol Biomarkers Prev . 1992;1:455–461. 28. Lu SH, Ohshima H, Fu HM. Urinary excretion of N-nitrosoamino acids and nitrate by inhabitants of high- and low-risk areas for esophageal cancer in Northern China: endogenous formation of nitrosoproline and its inhibition by vitamin C. Cancer Res . 1986;46:1485–1491. 29. Wu Y, Chen J, Ohshima H, et al. Geographic association between urinary excretion of N-Nitroso compounds and oesophageal cancer mortality in China. Int J Cancer. 1993;54:713–719. 30. Kamiyama S, Ohshima H, Shimada A, et al. Urinary excretion of N-nitrosoamino acids and nitrate by inhabitants in high- and low-risk areas for stomach cancer in northern Japan. IARC Sci Publ. 1987;84:479–502. 31. Ward MH, deKok TM, Levallois P, et al. Drinking water nitrate and health: recent findings and research needs. Environ Health Perspect

. 2005;113:1607–1614. 32. National Research Council. The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds. Washington, DC: National Academy Press; 1981. 33. National Research Council. Nitrate and Nitrite in Drinking Water. Washington, DC: National Academy Press; 1995.34. Bartsch H, Ohshima H, Pignatelli B. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention. Mutat Res . 1988;202:307–324. 35. Boeing H. Epidemiological research in stomach cancer: progress over the last ten years. J Cancer Res Clin Oncol. 1991;117:133–143. 36. Forman D. Dietary exposure to N-nitroso compounds and the risk of human cancer. Cancer Surv . 1987;6:719–738. 37. Chilvers C, Inskip H, Caygill C, et al. A survey of dietary nitrate in well-water users. Int J Epidemiol. 1984;13:324–331. Ward et al Epidemiology • Volume 17, Number 4, July 2006

© 2006 Lippincott Williams & Wilkins 

### 382