1 Public Health Sciences Division, Fred Hutchinson Cancer Research Center, PO Box 19024, Mail Stop M4-C308, Seattle, WA 98109-1024, USA
2 Epidemiology Department, School of Public Health and Community Medicine, University of Washington, Seattle, WA, USA
3 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
4 Department of Epidemiology and Biostatistics, University of California San Francisco, CA, USA
5 INSERM, U435, Rennes, France
6 Neuroepidemiology Research Unit, Istituto Nazionale Neurologico C Besta, Milan, Italy
7 Unidad de Información y Documentacion Medicosanitaria, IEDHC (CSIC-Universitat de Valencia), Valencia, Spain
8 Manitoba Cancer Treatment and Research Foundation, Winnipeg, Canada (deceased)
Correspondence: Dr Mueller, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, PO Box 19024, Mail Stop M4-C308, Seattle, WA 981091024, USA. E-mail: bmueller{at}fhcrc.org
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Abstract |
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Methods We used data from a multicentre, case-control study with maternal information on residential water source, and nitrate/nitrite levels of tap water measured by dipstick. Subjects included 836 CBT cases and 1485 controls from five countries.
Results The risks of CBT associated with reliance on well water (versus public water) during pregnancy varied widely, with significantly increased risks noted in two (of seven) regions and a decreased risk observed in one region. CBT risk did not increase with increasing nitrate levels. However, our results based on tap water tested in the pregnancy residences suggest the risk of astrocytoma may be associated with increasing levels of nitrite (odds ratio [OR] = 4.3, 95% CI: 1.4, 12.6 for nitrite levels of 1<5 mg/l nitrite ion; OR = 5.7, 95% CI: 1.2, 27.2 of nitrite 5 mg/l).
Conclusions These results should be interpreted with caution because women's recollection of water sources may have contained inaccuracies, and nitrate and nitrite measurements, available for only a portion of subjects, were often obtained years after the pregnancies occurred. However, our results suggest a need for closer evaluation of well water content in some regions and the possibility that a nitrite-related water exposure may be associated with CBT.
Accepted 30 March 2004
Nitroso compounds (NNC) have been evaluated in studies of environmental and dietary exposures and childhood brain tumours (CBT) because animal studies indicate nitrosoureas can induce brain tumours in offspring transplacentally.1 Drinking water is one possible source of exposure to nitrates, or possibly other chemicals with carcinogenic potential. Information about residential water source during pregnancy was obtained from the mothers of CBT cases and controls as part of an international collaborative case-control study of risk factors for CBT. In addition, residential tap water was tested using dipsticks for the presence of nitrates and nitrites. The purpose of this analysis was to compare the reported residential water sources among CBT cases and controls, and the presence of these chemicals in their water.
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Methods |
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Questions about the household water source from the period of time beginning one month prior to the pregnancy through the child's first year of life attempted to ascertain as many as two different sources of household water (public water, well, spring, rainwater/cistern, street tank, river, pond/lake, other) at each residence during that time. Because few women reported using sources other than public water, these were categorized as public water, wells, spring water, and other. Although public water may originate from many sources, women who reported using wells were likely to have relied on a non-public water source that may not have been monitored as closely by health departments in each region. Women also estimated the proportion of bottled water used at each residence.
The primary residential water source during the month prior to the pregnancy was identified and used as a measure of water source during the peri-conception month. Most women (91%) also had lived in this same house at the time of the child's birth. For the remaining women, we were unable to identify with certainty the water source(s) of the birth residence, or at the first month of pregnancy, because we lacked information regarding the specific months when women changed residences.
An additional water source at the peri-conception residence was reported by 28 women. Variables for any use of each type of water source were created to capture that information. Finally, all water sources reported by subjects were examined and variables created to indicate sole use of any single water source during the entire residential history.
For some subjects, interviewers also conducted a dipstick (Merck®, Darmstadt, Germany) measurement of tap water nitrates and nitrites. Although interviewers attempted to conduct this measurement for all respondents who lived in the same homes they had lived in during their pregnancies with the index children, many measurements were obtained from the current, rather than the index residence, and even if women still lived in the index residence, a measurement may not have been obtained. Dipstick measurements were obtained for 35% of all subjects (approximately 25% of those in all regions except Spain, at 62%), or 86% of those residing in their index residence (ranging from 72% in France, to 92% in Spain). Analyses of nitrate and nitrite measurements were conducted first for all dipstick measurements, and subsequently refined to include only those from the pregnancy residence, our best proxy measurement of household tap water exposure to nitrates during the mothers' pregnancies.
Information about residential water source and use of bottled water during pregnancy was not collected in Israel or Australia, and dipstick measurements were not obtained in Italy, Israel, or Australia. Our analyses based on water source therefore used data from the 836 cases and 1485 controls who lived in the participating regions. Analyses of dipstick measurements of tap water nitrates is restricted to the 283 cases and 537 controls with this information. Multivariable logistic regression4 was used to calculate odds ratio (OR) estimates and 95% CI for the relative risk of CBT associated with water source or nitrate/nitrite measures. As control subjects in some centres were individually matched on age and sex (and region in five centres), whereas at other centres frequency matching on age and sex was utilized, conditional logistic regression was initially performed with post hoc creation of individually linked case-control units. Subsequently, unconditional regressions stratified by centre, age, and sex were performed and are presented here, as the results were not altered by this approach, which also minimized the loss of data due to unmatched cases in regions where frequency matching was conducted. Results presented are from analyses restricted to subjects with known information for all variables included in the models.
Other factors, in addition to centre, sex, and age at diagnosis, that were considered for their potential effects on the relationships of interest included diagnosis year, maternal prenatal smoking, and mother's educational level. Only factors that meaningfully altered the risk estimates were retained in the analysis. Unless otherwise indicated, all risk estimates are adjusted for age, sex, and diagnosis year. Tumour type-specific analyses were conducted with cases classified5 as astroglial (93809384, 94009421, 94249442), primitive neuroectodermal tumours (PNET) (94709473; 9501), and all other tumours.
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Results |
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Tap water nitrate and nitrite
When all dipstick measurements were included, 63% of cases and 56% of controls had nitrate levels below the level of detection (<10 mg/l nitrate ion) in their residential tap water, and the presence of nitrate at any level was not associated with an increased risk of CBT (Table 3). The OR for nitrate levels 10 mg/l were consistently, although not significantly, below one. The presence of detectable nitrite (>1 mg/l nitrite ion) at any level was not associated with an increased risk of CBT, with risk estimates of 0.6 (95% CI: 0.4, 1.1) for levels between 1<5 mg/l, and 0.8 (95% CI: 0.4, 1.9) for levels >5 mg/l. Restriction of these analyses to women (207 cases, 400 controls) who reported they had not used bottled water during their pregnancies did not meaningfully alter the OR.
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When histological tumour group-specific analyses were conducted, an increased risk of astroglial tumours only was associated with measurable nitrite levels at the pregnancy residence (OR = 4.3, 95% CI: 1.4, 12.6 for 1<5 mg/l nitrite; OR = 5.7, 95% CI: 1.2, 27 for 5 mg/l nitrite, Table 4). This pattern was also observed when results were stratified by children's ages at diagnosis (data not shown). We observed no increased risks of tumours within the PNET group, or other tumour type group. Due to the limited numbers available, we were unable to further restrict this analysis to only those who did not use bottled water.
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Discussion |
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Even if women accurately recalled their water source, community supply drinking water in many areas generally is a combination of multiple sources, and thus our reference category of public supply water is likely a heterogeneous mix of waters from many sources and/or locations. Common factors most likely distinguishing public supply from well water are routine disinfection procedures (with potential for by-products) and relative level of monitoring by public health agencies. Private wells may be tested less frequently than are municipal water supplies for nitrates, pesticides, and other contaminants, some of which have been hypothesized to be associated with childhood cancer.6 Our dipstick data, though limited in numbers, lend some support to this possibility; 33% of the 24 pregnancy residences relying on wells, versus 11% of those reporting other water sources (n = 500), had nitrate levels >25 mg/l, and 4% versus 3% had nitrite levels of 5 mg/l.
Some results from the three US regions7 and France8 related to water source have been reported previously, although results related to peri-conception source, and for well versus public supply water as sole sources have not been previously presented. The reason for the decreased risk of CBT associated with well water use observed in the Los Angeles region is unclear. Wells as a sole source of household water were infrequently reported (by 20/619 respondents) in largely urban Los Angeles County, and it is possible these households differed from others in that region in ways that we could not measure.
Pregnant women who drink nitrate-contaminated water may be at increased risk of having spontaneous abortions9 or giving birth to infants with congenital malformations, especially of the central nervous system.1012 Although the role of nitrate in drinking water as a risk for cancer is controversial,13 high levels of nitrate in drinking water have been associated with increased mortality1416 and incidence1721 of some cancers and lesions. However, negative results have also been reported for some cancer sites.20,21 One case-control study of adult brain tumours in Germany, which used dipsticks to measure nitrate levels in addition to municipal water records, did not observe increased risks of brain tumours associated with either measurable nitrates as assessed using dipsticks, or by mean nitrate levels in community water supplies.22
The mechanism by which dietary nitrates may form potentially carcinogenic NNC involves endogenous reduction into nitrite, and subsequent nitrosation of amines and amides into potentially carcinogenic chemicals.23 Although drinking water represents only a fraction of total dietary nitrate, it represents 70% of dietary nitrate intake among individuals with >50 mg/l nitrate ion in their drinking water.24 Unlike vegetables (the other major source of dietary nitrate), drinking water does not contain vitamins that inhibit endogenous formation of NNC.25 There is evidence of a doseresponse increase in urinary nitrate secretion,26 and of increased salivary nitrate and nitrite levels among those exposed to higher levels of drinking water nitrates, relative to those with low levels,27 but the potential carcinogenic effect of this is unclear. Increased occurrence of sister chromatid exchange was not associated with relatively high drinking water nitrate levels.26 However, an increased HPRT variant frequency in peripheral lymphocytes suggestive of genotoxic exposure was associated with ingestion of high levels of drinking water nitrates.27 As pregnant women drink more water,28,29 and bathe or shower more frequently than non-pregnant women,29 their exposure to water contaminants via consumption, vaporization, and dermal absorption is increased. In addition, the fetal brain may be particularly vulnerable to environmental exposures.30
A limitation affecting both self-reported water source information and dipstick measurements is our inability to take into consideration (aside from excluding bottled water users) individual tap water consumption levels. Our results related to the dipstick measurements were not affected by recall bias or other limitations of self-reported data, but there are limitations. Dipstick measurements were available for only 28% of all subjects and the possibility of selection bias must be considered. Overall, 32% of subjects resided in the same homes in which they lived during the index pregnancy (24% to 32% in all regions except Spain, at 64%). Although we obtained dipstick measurements from 86% of these homes (ranging from 72% in France to 98% in Spain), and the fact that similar proportions of cases (30%) and controls (33%) resided in their pregnancy residences, even our analyses restricted to pregnancy residence may be biased if there are case-control differences in residential mobility patterns. Another limitation is that dipstick measurements were conducted, in many instances, several years after women's pregnancies. Even if the residences remained the same, the water tested may not represent the same content as during the women's index pregnancies. Other limitations are the semi-quantitative nature of the dipsticks, possible differences in interpretation of results among raters, and the possibility that, despite our attempt to adhere to the manufacturer's instructions, some interferences and inaccuracies may have occurred in their use. One concern is that the presence of nitrite in a water sample may interfere with the nitrate reading, due to the reaction method employed by the dipstick. Because of this, measurements for samples containing both nitrite and nitrate may overestimate the nitrate level actually present. Given the semi-quantitative nature of the dipsticks, we were unable to devise any suitable adjustment of nitrate readings when both chemicals were present. However, when analyses were restricted to households with nitrites below the level of detection, the CBT risk estimates related to nitrate level did not change. As the presence of nitrates in a sample does not affect the nitrite measurements, no similar interference would have affected our nitrite measurements.
The maximum allowable nitrate level for drinking water under the World Health Organization and European Union maximum admissible concentration standards is 50 mg/l nitrate ion. (The United States Environmental Protection Agency [EPA] drinking water standard is 10 mg/l as nitrate-nitrogen, roughly equivalent to 45 mg/l nitrate ion.) When all tested residences were included, we did not observe an increased risk of CBT associated with measurable levels of nitrate. In fact the risk estimates for the lowest levels of measurable nitrate were fairly consistently less than one, although not statistically significant. However, when analyses were conducted by tumour type the risk of astroglial tumours associated with nitrate levels 50 mg/l were modestly, but not significantly increased. Our data suggest that high levels of nitrite in residential water consumed during pregnancy may be associated with an increased risk of CBT, in particular, for astroglial tumours. More complete ascertainment of prenatal exposure may help address this in future studies.
Homes with wells, or with higher tap water nitrate or nitrite levels, may be more likely to be located in or near agricultural areas. Parental employment in agriculture,31 and exposure to farm animals32 has been associated with CBT in our data and in other studies.33 In our data, the small numbers of mothers who lived or worked on farms during their pregnancies limited our ability to examine this in each region. Finally, the presence of high levels of nitrate in wells is associated with the presence of other contaminants, including pesticides and metals,34 which were not measured in this study. It is possible that the nitriteCBT association we observed in water tested at the pregnancy residence among non-bottled water users is due to an unmeasured nitrite-related factor or contaminant. The fact that the relationships we observed were most marked, or present only among those for whom these household drinking water exposures may be greatest, suggest that regardless of the aetiological factor(s), the prenatal window may be particularly important for exposures related to drinking water contaminants.
KEY MESSAGES
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Acknowledgments |
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This work was part of an international collaborative study of childhood brain tumours co-ordinated by the SEARCH Program of the International Agency for Research on Cancer. The authors thank the other collaborators who gave valuable input, particularly during the design of the study and the development of the study questions. Additionally, the authors thank Ann Hadley, Tina Paoff, Maria Paul, Isabel Gaeta, Katherine Newton, Barbara Nist, Jennifer Kristiansen, and Susan Parrisher for their contributions to this work.
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