Open Access

Research Article

Nitrate Levels in Groundwater and Water Supplies Top

Nitrate is the most common chemical contaminant in the world’s groundwater aquifers (Spalding and Exner 1993). An estimated 42% of the U.S. population uses groundwater as their drinking-water supply (Hutson et al. 2004). In the United States, total nitrogen in streams and nitrate in groundwater are highest in agricultural areas, followed by urban areas and areas with mixed land use (Figure 1). The most recent data indicate that about 22% of domestic wells in agricultural areas of the United States exceeded the MCL (U.S. Geological Survey, unpublished data). In contrast, 3% of public supply wells in major aquifers (typical sources for public water supplies) exceed the MCL (U.S. Geological Survey, unpublished data).

The exposure picture is similar in the European Union. Public water supplies are largely below the WHO guideline; however, in some countries, private wells in rural areas have elevated nitrate concentrations reaching 10–15 times the recommended level (European Environment Agency 2003). Overall, nitrate levels exceeded the guideline in about one-third of the groundwater bodies for which data were available (European Environment Agency 2003). Several eastern European countries report high levels of nitrate contamination in a large proportion of private wells; for example, in Romania, 20% of 2,000 wells had nitrate levels > 23 mg/L as nitrate-N (Jedrychowski et al. 1997). Studies from other countries, including China, Botswana, Turkey, Senegal, and Mexico, report private well water levels that exceed the WHO guideline, in some instances at levels > 68 mg/L nitrate-N (WHO 2004a). Fertilizer is the main contributing factor in agricultural areas; however, nitrogen from human waste appears to be the most important source in urban areas lacking centralized water and sanitation systems. Although systematic information on nitrate levels in groundwater in other parts of the world is more limited, empirical modeling approaches have indicated that users of shallow wells in areas with high nitrogen inputs, well-drained soils, and unconsolidated rocks are most at risk of consuming high-nitrate groundwater (Nolan et al. 2002).

Methemoglobinemia Top

Ingested nitrate is reduced to nitrite, which binds to hemoglobin to form methemoglobin (MetHb). Methemoglobinemia occurs when elevated levels of MetHb (exceeding about 10%) interfere with the oxygen-carrying capacity of the blood. Infants are particularly susceptible to developing methemoglobinemia for several reasons, including their increased capacity to convert nitrate to nitrite and their lower levels of the enzyme cytochrome b5 reductase, which converts MetHb back to hemoglobin. Methemoglobinemia in infants fed formula made with well water with high nitrate levels was first reported in 1945 by Comly (1945). The regulatory level for nitrate in drinking-water supplies was determined after a survey of infant methemoglobinemia case reports in the United States indicated that no cases were observed at drinking-water nitrate levels < 10 mg/L nitrate-N (Walton 1951). Because an estimated 22% of domestic wells in agricultural regions of the United States exceed the nitrate MCL (U.S. Geological Survey, unpublished data), it is likely that significant numbers of infants are given water containing > 10 mg/L nitrate-N. Nevertheless, few cases of methemoglobinemia have been reported since the MCL was promulgated.

The risk of methemoglobinemia among infants depends on many factors other than the ingestion of nitrate in drinking water. Some foods and medications contain high levels of nitrate (Sanchez-Echaniz et al. 2001). Enteric infections, potentially caused by fecal bacteria contamination in wells, may lead to the endogenous production of nitrite, as evidenced by numerous published reports of infants with diarrhea and methemoglobinemia but no apparent exposure to exogenous MetHb-forming agents (Charmandari et al. 2001; Hanukoglu and Danon 1996; Levine et al. 1998; Wennmalm et al. 1993). The consumption of antioxidants such as vitamin C appears to be a protective factor. Finally, polymorphisms in the activity of cytochrome b5 reductase may mediate the effect of ingested nitrate or endogenously produced nitrite (Gupta et al. 1999).

Studies that have examined the relationship between nitrate levels in drinking water and MetHb levels in infants have produced mixed results (U.S. EPA 1991). The few experimental studies are largely negative; however, most of these studies evaluated low levels of drinking-water nitrate and included few infants. Cofactors such as diarrhea and respiratory diseases reportedly increase MetHb levels (Shearer et al. 1972; Shuval and Gruener 1972). An epidemiologic study in South Africa (Super et al. 1981) found an increase in MetHb levels in infants fed water with nitrate > 20 mg/L nitrate-N; however, clinical methemoglobinemia was rarely found. A protective effect of vitamin C intake on MetHb was noted (Super et al. 1981). More recently, a retrospective, nested case–control study in Romania found an association between nitrate exposure from drinking water and clinical methemoglobinemia, but also some evidence of an association with diarrheal disease (Zeman et al. 2002). Gupta and colleagues (1999) found cytochrome b5 reductase activities to be higher among those consuming water with high nitrate levels, indicating a level of adaptation to the consumption of high nitrate waters.

Recently, the role of nitrate exposure alone in causing methemoglobinemia has been questioned (Avery 1999; Fewtrell 2004; Hanukoglu and Danon 1996). Clearly, we need to better understand the interaction of factors that lead to methemoglobinemia to assess the relative importance of each factor and to identify the conditions under which exposure to nitrate in drinking water poses a risk of methemoglobinemia.

Nitrate Intake and Endogenous Formation of N-Nitroso Compounds Top

Nitrate is a precursor in the formation of N-nitroso compounds (NOC), a class of genotoxic compounds, most of which are animal carcinogens. In the human body, nitrate is a stable, inert compound that cannot be metabolized by human enzymes. However, the nitrate-reducing activity of commensal bacteria may convert nitrate into nitrite and other bioactive nitrogen compounds that affect physiological processes and human health. After ingestion, nitrate is readily absorbed from the upper gastrointestinal tract. Up to 25% is actively excreted in saliva, where about 20% is converted to nitrite by bacteria in the mouth (Spiegelhalter et al. 1976). This conversion can occur at other sites including the distal small intestine and the colon.

Under acidic conditions in the stomach, nitrite is protonated to nitrous acid (HNO₂), which in turn spontaneously yields dinitrogen trioxide (N₂O₃), nitric oxide (NO), and nitrogen dioxide (NO₂). NO is a bioactive compound known to play a role in vasodilatation and in defense against periodontal bacteria and other pathogens. N₂O₃, on the other hand, is a powerful nitrosating agent capable of donating NO⁺ to secondary and tertiary amines to form potentially carcinogenic N-nitrosamines (Leaf et al. 1989). Alternatively, HNO₂ can be protonated to H₂NO₂, which reacts with amides to form N-nitrosamides. At neutral pH, nitrite can be reduced by bacterial activity to form NO, which can react with molecular oxygen to form the nitrosating compounds N₂O₃ and nitrogen tetroxide (N₂O₄). In addition to the acid-catalyzed and bacterial-catalyzed formation of nitrosating agents, inducible NO synthase activity of inflammatory cells can also produce NO (Ohshima and Bartsch 1994). Together, these three mechanisms of endogenous nitrosation account for an estimated 40–75% of the total human exposure to NOC (Tricker 1997). Other sources of human exposure include pre-formed NOC found in preserved meats and fish, beer, certain occupational exposures, and tobacco products (Tricker 1997).

Several studies support a direct relationship between nitrate intake and endogenous formation of NOC. High intake of drinking-water nitrate (above the MCL) is associated with an increased endogenous capacity to nitrosate proline (Mirvish et al. 1992; Moller et al. 1989). In addition, populations with high rates of esophageal and gastric cancer excrete high levels of N-nitrosoproline (Kamiyama et al. 1987; Lu et al. 1986). Nitrate intake at the acceptable daily intake level (3.67 mg/kg body weight, 0.84 mg/kg as nitrate-N) results in increased urinary excretion of NOC, particularly in combination with increased intake of dietary nitrosatable precursors (Vermeer et al. 1998). However, a Canadian population exposed to nitrate below the acceptable daily intake level showed no relationship between nitrate levels in drinking water and urinary nitrosamines (Levallois et al. 2000).

Factors that modulate endogenous nitrosation.

Although intake of high drinking-water nitrate is consistently associated with endogenous nitrosation capacity, intake of dietary nitrate is less likely to increase nitrosation, because of the presence of nitrosation inhibitors in vegetables, the major contributors to dietary nitrate intake (Bartsch et al. 1988; National Academy of Sciences 1981). Dietary compounds that inhibit endogenous nitrosation include vitamin C, which has the capacity to reduce HNO₂ to NO, and alphatocopherol, which can reduce nitrite to NO. Several epidemiologic studies reported no association or inverse associations between dietary nitrate intake and human cancers (Boeing 1991; Forman 1987; Ward et al. 1996), which may be because of the antioxidants and nitrosation inhibitors in nitrate-containing foods (Bartsch et al. 1988). Inhibitory effects on nitrosation have also been described with betel nut extracts, ferulic and caffeic acid, garlic, coffee, and green tea polyphenols (Stich et al. 1984). In addition, nondietary factors such as the use of mouthwashes containing chlorhexidine can influence the endogenous nitrosating capacity (van Maanen et al. 1998).

Apart from the level of nitrosating agents, the level of nitrosatable precursors in the diet, which come predominantly from meat and fish, is a crucial factor in endogenous nitrosation. Dietary intakes of red and processed meat are of particular importance in the formation of fecal NOC (Bingham 1999; Bingham et al. 1996, 2002; Cross et al. 2003; Haorah et al. 2001). Higher consumption of red meat (600 vs. 60 g/day), but not white meat, resulted in a 3-fold increase in fecal NOC levels (Bingham et al. 1996). Colon cancer incidence is most consistently associated with consumption of red meat (beef, lamb, and pork), but not with poultry and fish (Bingham 1999). Dietary supplementation of a diet low in red meat with either heme iron or inorganic iron demonstrated that heme in particular was able to stimulate endogenous nitrosation (Cross et al. 2003), thereby providing a possible explanation for the differences in colon cancer risk between red and white meat consumption. Additionally, this linkage may be stronger for processed meat than for fresh meat because of the higher NOC and NOC precursor levels in processed meat.

Endogenous nitrosation can also be stimulated by inflammatory and other medical conditions. For instance, patients with bilharzia have an increased bladder cancer risk associated with increased urinary levels of nitrite and volatile nitrosamines, most likely generated by the reaction of inflammation-derived NO with amines present in the urine (Tricker et al. 1989). Inflammatory bowel disease is also related to both increased nitro-sation and cancer risk (Lashner et al. 1988). During inflammatory bowel disease, increased inducible NO synthase activity can produce excess NO, which is oxidized to nitrogen oxides and nitrite, which in turn react with nitrosatable precursors in colonic contents to produce NOC. Indeed, ulcerative colitis patients showed increased levels of inducible NO synthase in the colonic mucosa (Kimura et al. 1998) and of NO and nitrite in the colonic lumen (Lundberg et al. 1997; Roediger et al. 1990). Increased levels of fecal NOC have been found in patients with inflammatory bowel disease and in mice with chemically induced colitis (de Kok et al. 2005; Mirvish et al. 2003).

Health Effects Associated with Drinking-Water Nitrate Top

Cancer.

NOC are potent animal carcinogens, inducing tumors at multiple organ sites including the esophagus, stomach, colon, bladder, lympatics, and hematopoietic system (Bogovski and Bogovski 1981). NOC cause tumors in every animal species tested, and it is unlikely that humans are unaffected (Lijinsky 1986). The number of well-designed epidemiologic studies with individual exposure data and information on nitrosation inhibitors and precursors are few for any single cancer site, limiting the ability to draw conclusions about cancer risk.

Most studies have been ecologic in design, linking incidence or mortality rates to drinking-water nitrate levels at the town or county level. The early studies focused on stomach cancer mortality, and most used drinking-water nitrate measurements concurrent with the period of cancer mortality. Results were mixed, with some studies showing positive associations, many showing no association, and a few showing inverse associations (Cantor 1997). Recent ecologic studies of stomach cancer in Slovakia, Spain, and Hungary with historical measurements and exposure levels near or above the MCL have found positive correlations with stomach cancer incidence or mortality (Gulis et al. 2002; Morales-Suarez-Varela et al. 1995; Sandor et al. 2001). Two studies included other cancer sites. In Slovakia, incidence of non-Hodgkin lymphoma (NHL) and colon cancer was significantly elevated among men and women exposed to public supply nitrate levels of 4.5–11.3 mg/L nitrate-N (Gulis et al. 2002); there was no association with bladder and kidney cancer incidence. In Spain, there was a positive correlation between nitrate levels in public supplies and prostate cancer mortality, but no relation with bladder and colon cancer (Morales-Suarez-Varela et al. 1995).

In the past decade, several case–control and cohort studies have evaluated historical nitrate levels in public water supplies (largely < 10 mg/L nitrate-N) and risk of several cancers (Table 1). Some studies evaluated factors affecting nitrosation, such as vitamin C intake. A cohort study of older women in Iowa (USA) (Weyer et al. 2001) found a 2.8-fold and 1.8-fold risk of bladder and ovarian cancers, respectively, associated with the highest quartile (> 2.46 mg/L nitrate-N) of the long-term average nitrate levels at the current residence. They observed significant inverse associations for uterine and rectal cancer and no significant associations for NHL, leukemia, colon, rectum, pancreas, kidney, lung, and melanoma. Case–control studies of bladder (Ward et al. 2003), brain (Ward et al. 2004), colon and rectum (De Roos et al. 2003), and pancreas cancer (Coss et al. 2004) in Iowa found no association between cancer risk and average nitrate levels over almost 30 years. Each study evaluated the interaction between nitrosation inhibitors or NOC precursors and nitrate intake from drinking water. For colon cancer, there was a significant positive interaction between 10 or more years of exposure above 5 mg/L nitrate-N and both low vitamin C and high meat intake, factors likely to increase endogenous NOC formation (De Roos et al. 2003).

A case–control study of NHL in Nebraska (USA) (Ward et al. 1996) found a significant positive association between the average nitrate level in public water supplies over about 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, a recent study of NHL in Iowa with similar exposure levels found no association (Ward et al. 2004). A case–control study of NHL in Minnesota (USA) (Freedman et al. 2000) with lower levels of nitrate found an inverse association among those with the highest level (> 1.5 mg/L nitrate-N). Case–control studies in Nebraska (Ward et al. 2004) and Germany (Steindorf et al. 1994) found no association with long-term average nitrate levels in public water supplies and adult brain cancer. The Nebraska study found no evidence of an interaction with vitamin C intake. A case–cohort analysis of stomach cancer within a cohort study in the Netherlands (van Loon et al. 1998) found no association with quintiles of water nitrate intake determined from public supply levels.

Specific NOC are transplacental neurocarcinogens in animal studies. A study of childhood brain cancer measured nitrate levels in water supplies using dipstick measurements, often many years after the pregnancy (Mueller et al. 2001). Measured levels of nitrate and nitrite were not associated with risk; however, women in western Washington State, one of the three study centers, who used private wells as their drinking-water source during the pregnancy had a significantly increased risk of brain cancer in their offspring.

Adverse reproductive outcomes.

In 1961, Schmitz described a possible relationship between high maternal MetHb levels and spontaneous abortion. Since then, at least 10 studies have examined the association between drinking-water nitrate and adverse reproductive outcomes. Table 2 summarizes these studies by location, study design, determination of water nitrate, and key findings. Few studies have been published regarding water nitrate and the outcomes of spontaneous abortions, stillbirths, premature birth, or intrauterine growth retardation. Results of these studies have been inconsistent, possibly indicating no true effect of water nitrate on reproductive outcomes at the levels evaluated in these studies. Alternatively, the inconsistencies may be due to the differing periods over which exposure was assessed, differing levels of water nitrate across studies, or differences in exposure to other cofactors.

Results of studies evaluating drinking-water nitrate and congenital malformations in offspring are also mixed (Table 2). Four studies (Arbuckle et al. 1988; Brender et al. 2004a, 2004b; Croen et al. 2001, Dorsch et al. 1984) found positive associations between drinking-water nitrate and congenital malformations, particularly malformations of the central nervous system, and specifically neural tube defects (NTDs). In each of these studies, water nitrate levels associated with increased risk of these defects were below the MCL, although the 95% confidence intervals (CIs) for some of the risk estimates were consistent with unity and varied by the source of water (groundwater, mixed, or surface). Two of these studies (Brender et al. 2004b; Croen et al. 2001) also examined dietary intake of nitrates and nitrates and NTDs and found minimal or no effect on risk. In a study of nitrosatable drug exposure and risk of NTDs (Brender et al. 2004b), drinking-water nitrates and dietary nitrites/total nitrites substantially modified the risk associated with this drug exposure during the periconceptional period; higher levels of nitrates in food or water significantly increased the risk of NTDs if women were exposed to such drugs.

Other health outcomes.

Animal studies suggest that nitrate at high doses can competitively inhibit iodine uptake and induce hypertrophic changes in the thyroid (Bloomfield et al. 1961). In a human biomonitoring study in the Netherlands, consumption of water with nitrate levels at or above the MCL was associated with thyroid hypertrophy (van Maanen et al. 1994) and genotoxic effects (van Maanen et al. 1996). Animal studies provide evidence that NOC can damage the pancreatic beta cells (Longnecker and Daniels 2001). Three epidemiologic studies (Kostraba et al. 1992; Parslow et al. 1997; van Maanen et al. 2000) that were ecologic in design found a positive correlation between drinking-water nitrate levels below the MCL and the incidence of type I childhood diabetes, although the association observed by van Maanen was not statistically significant. Other studies have found associations between water nitrate exposure and increased blood pressure (Pomeranz et al. 2000) and acute respiratory tract infections in children (Gupta et al. 2000).

Recommendations for Future Research Top

Experimental/human biomonitoring studies.

Endogenous nitrosation in humans has been demonstrated in relation to drinking-water nitrate ingestion at levels above the MCL. However, further studies are needed to determine the extent of endogenous nitrosation at intermediate drinking-water nitrate levels (5–10 mg/L as nitrate-N) and to clarify the role of nitrate from water versus food sources. Furthermore, the role of precursors and modulators of NOC formation should be more fully investigated. These future studies should be conducted among healthy individuals as well as individuals with medical conditions that increase endogenous nitrosation.

In view of the complex kinetics of NOC formation and the organ specificity of several of these compounds (Hodgson et al. 1980; Suzuki et al. 1999), more studies are needed to evaluate the relationship between nitrate intake and formation, metabolism, and excretion of NOC. Ideally, a physiologically based pharmacokinetic model should be developed as previously recommended (National Research Council 1995) to predict exposure to NOC from all sources of nitrate exposure (exogenous and endogenous), nitrite intake, the transformation of nitrate into nitrite, and antioxidant intake. However, this will require additional data on the formation of individual NOC as well as their respective toxicologic characteristics. The results of these investigations will reveal the value of different markers of NOC exposure in future epidemiologic studies. Future studies linking NOC exposure to early markers of effect or to the actual disease will clarify the role of endogenous nitrosation and NOC exposure as etiologic factors.

Because many NOC require α -hydroxylation by CYP2E1 for bioactivation and for formation of DNA adducts, it is important to investigate the influence of polymorphisms in the gene encoding for this enzyme. One study found that specific variants in this gene are linked to increased rectum cancer risk, particularly in subjects with high intake of red and processed meat, who are exposed to increased levels of NOC (Le Marchand et al. 2002). Moreover, gene expression levels of human CYP2E1 were related to cytotoxicity and DNA damage by nitrosamines in pancreatic beta-cell lines, suggesting that such gene environment interactions are also relevant in type 1 diabetes (Lees Murdock et al. 2004). These promising lines of research point to a possible interaction between drinking-water nitrate exposure and gene expression of and/or genetic variation in CYP2E1, which may also influence the risk of several adverse health outcomes associated with nitrate exposure.

Epidemiologic studies.

Methods must be developed and validated to improve estimates of current and historical exposure to nitrate via food and water, particularly for populations served by private wells, which are less likely to be routinely monitored. Future epidemiologic studies should integrate a) exposure assessment for nitrate intake from drinking water, nitrate and nitrite intake from the diet, and amines and amides from dietary and drug sources, b) endogenous exposure to NOC by analysis of relevant biological media (e.g., saliva, urine, feces), and c) reliable health risk markers (e.g., biomarkers of genotoxicity) or diagnosis of actual disease.

Future studies should include populations with well-characterized long-term exposures, including those who use private wells, which can have higher nitrate levels than public supplies. With the increasing availability of public water supply monitoring data (many U.S. states have almost 40 years of measurements), further detailed exposure assessment of populations using public supplies is also feasible. Drinking-water contaminants that may occur along with nitrate, such as agricultural pesticides, should also be evaluated. Geographic-based modeling efforts to predict the probability of high nitrate concentrations in groundwater, using information on nitrogen inputs from agricultural and urban sources (Nolan et al. 2002), is a promising approach for estimating drinking-water nitrate exposure for the population using private wells.

Additional studies of drinking-water nitrate and cancer are needed to follow up on the suggestive positive findings to date and to evaluate other cancer sites in which endogenously formed NOC may play a role. Studies of reproductive outcomes should address the exposure period most relevant for the specific outcome of interest. Maternal residential mobility between conception and birth may lead to misclassification of exposure if the water source at birth is used in studies of spontaneous abortions and congenital malformations. Studies must be of sufficient size to allow for examination of specific defects rather than groups of defects by system, because combining different defects might mask associations. More research is needed on the relation between water nitrate and the reproductive outcomes of spontaneous abortion, fetal death, premature birth, and intrauterine growth retardation.

In the design and analysis stage, future epidemiologic studies should consider factors that modulate endogenous nitrosation, as discussed above, to be able to evaluate potential interactions of water nitrate intake with these factors, thus providing stronger evidence for or against an association. In particular, studies of susceptible populations may be fruitful, and epidemiologic studies should be designed with sufficient power to evaluate risk among potentially susceptible subgroups. Such populations include patients with different forms of chronic inflammation (such as inflammatory bowel disease), patients infected with nitrate-reducing bacteria (such as in periodontal disease), those with low intake of vitamins and other known nitrosation inhibitors, or those with a history of high incidence of potentially NOC-related diseases. The people of Linxian County in China, for example, are known for their persistently low intake of several micronutrients and high risk of esophageal cancer (Blot et al. 1993). Such populations will likely benefit from preventive measures taken as a result of these investigations.

Conclusions Top

Adverse health effects from drinking-water nitrates are most likely the result of a complex interaction of the amount of nitrate ingested, the concomitant ingestion of nitrosating cofactors and precursors, and medical conditions of the host that may increase nitrosation. Furthermore, these effects may be attenuated by inhibitors of endogenous nitrosation such as vitamin C and alphatocopherol. We recommend that future studies take into account such complexities in understanding the relation between drinking-water nitrates and cancer, adverse reproductive outcomes, and other health outcomes.

Several authors (Avery 1999; L’hirondel and L’hirondel 2002) have questioned the importance of nitrate in drinking water as a risk factor for methemoglobinemia and have suggested that the current nitrate standard might be safely raised to 15–20 mg/L nitrate-N with no increase in methemoglobinemia cases. A better understanding of the conditions under which nitrate in drinking water poses a risk of methemoglobinemia is clearly needed, particularly in light of recent cases of methemoglobinemia associated with well water levels between 20 and 30 mg/L nitrate-N (Knobeloch et al. 2000). Most importantly, the role of nitrate as a risk factor for cancer and adverse reproductive outcomes must be more thoroughly explored before changes to nitrate water quality standards are considered.

Figures and Tables Top

Figure 1.

Interquartile range of total nitrogen in streams and nitrate-N in groundwater in agricultural, urban, and mixed land use, and undeveloped areas of the United States. Upper bound of bar represents 90th percentile and lower bound represents 10th percentile. Along the top of the graph are the number of stream sampling stations and groundwater networks (group of wells in an aquifer).

Table 1.

Analytic epidemiologic studies of drinking-water nitrate ^a and cancer.

Table 2.

Studies of the relation between drinking-water nitrate ^a and reproductive outcomes.

References Top

1. Arbuckle TE, Sherman GJ, Corey PN, Walters D, Lo B 1988. Water nitrates and CNS birth defects: a population-based case-control study Arch Environ Health 43:162–167.3377550 Find this article online
2. Aschengrau A, Zierler S, Cohen A 1989. Quality of community drinking water and the occurrence of spontaneous abortion Arch Environ Health 44:283–290.2554824 Find this article online
3. Aschengrau A, Zierler S, Cohen A 1993. Quality of community drinking water and the occurrence of late adverse pregnancy outcomes Arch Environ Health 48:105–113.8476301 Find this article online
4. Avery AA 1999. Infantile methemoglobinemia: reexamining the role of drinking water nitrates Environ Health Perspect 107:1–8.10068291 Find this article online
5. Bartsch H, Ohshima H, Pignatelli B 1988. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention Mutat Res 202:307–324.3057363 Find this article online
6. Bingham SA 1999. High-meat diets and cancer risk Proc Nutr Soc 58:243–248.10466162 Find this article online
7. Bingham SA, Hughes R, Cross AJ 2002. Effect of white versus red meat on endogenous N-nitrosation in the human colon and further evidence of a dose response J Nutr 132:3522S–3525S.12421881 Find this article online
8. Bingham SA, Pignatelli B, Pollock JR, Ellul A, Malaveille C, Gross G, et al. 1996. Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer? Carcinogenesis 17:515–523.8631138 Find this article online
9. Bloomfield RA, Welsch CW, Garner GB, Muhrer ME 1961. Effect of dietary nitrate on thyroid function Science 134:1690.13870154 Find this article online
10. Blot WJ, Li J, Taylor PR, Guo W, Dawsey S, et al. 1993. Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population J Natl Cancer Inst 85:1483–1491.8360931 Find this article online
11. Boeing H 1991. Epidemiological research in stomach cancer: progress over the last ten years J Cancer Res Clin Oncol 117:133–143.2036128 Find this article online
12. Bogovski P, Bogovski S 1981. Animal species in which N-nitroso compounds induce cancer Int J Cancer 27:471–474.7275353 Find this article online
13. Brender J, Olive J, Felkner M, Suarez L, Hendricks K, Marckwardt W 2004a. Intake of nitrates and nitrites and birth defects in offspring [Abstract] Epidemiology 15:S184. Find this article online
14. Brender JD, Olive JM, Felkner M, Suarez L, Marckwardt W, Hendricks KA 2004b. Dietary nitrites and nitrates, nitrosatable drugs, and neural tube defects Epidemiology 15:330–336. Find this article online
15. Bukowski J, Somers G, Bryanton J 2001. Agricultural contamination of groundwater as a possible risk factor for growth restriction or prematurity J Occup Environ Med 43:377–383.11322099 Find this article online
16. Cantor KP 1997. Drinking water and cancer Cancer Causes Control 8:292–308.9498894 Find this article online
17. Cedergren MI, Selbing AJ, Lofman O, Kallen BAJ 2002. Chlorination byproducts and nitrate in drinking water and risk for congenital cardiac defects Environ Res 89:124–130.12123645 Find this article online
18. Charmandari E, Meadows N, Patel M, Johnston A, Benjamin N 2001. Plasma nitrate concentrations in children with infectious and noninfectious diarrhea J Pediatr Gastroenterol Nutr 32:423–427.11396807 Find this article online
19. Comly H 1945. Cyanosis in infants caused by well water JAMA 129:112–116. Find this article online
20. Coss A, Lynch C, Cantor KP, Ward MH 2004. Pancreatic cancer and drinking water and dietary sources of nitrate and nitrite Am J Epidemiol 159:693–701.15033647 Find this article online
21. Croen LA, Todoroff K, Shaw GM 2001. Maternal exposure to nitrate from drinking water and diet and risk for neural tube defects Am J Epidemiol 153:325–331.11207149 Find this article online
22. Cross AJ, Pollock JR, Bingham SA 2003. Heam, not protein or inorganic iron, is responsible for endogenous intestinal N-nitrosation arising from red meat Cancer Res 63:2358–2360.12750250 Find this article online
23. De Kok TM, Engels LG, Moonen E, Kleinjans JC 2005. Inflammatory bowel disease stimulates formation of carcinogenic N-nitroso compounds Gut 54:731.15831929 Find this article online
24. DeRoos AJ, Ward MH, Lynch CF, Cantor KP 2003. Nitrate in public water systems and the risk of colon and rectum cancers Epidemiology 14:640–649.14569178 Find this article online
25. Dorsch MM, Scragg RKR, McMichael AJ, Baghurst PA, Dyer KF 1984. Congenital malformations and maternal drinking water supply in rural South Australia: a case-control study Am J Epidemiol 119:473–486.6711537 Find this article online
26. Ericson A, Kallen B, Lofkvist E 1988. Environmental factors in the etiology of neural tube defects: a negative study Environ Res 45:38–47.3338434 Find this article online
27. European Environment Agency 2003. Europe’s Environment: The Third Assessment. Environmental Assessment Report No. 10. Copenhagen:European Environment Agency
28. Fewtrell L 2004. Drinking-water nitrate, methemoglobinemia, and global burden of disease: a discussion Environ Health Perspect 112:1371–1374.15471727 Find this article online
29. Fields S 2004. Global nitrogen: cycling out of control Environ Health Perspect 112:A557–A563. Find this article online
30. Forman D 1987. Dietary exposure to N-nitroso compounds and the risk of human cancer Cancer Surv 6:719–738.3330686 Find this article online
31. Freedman DF, Cantor KP, Ward MH, Helzlsouer K 2000. Nitrates in drinking water and non-Hodgkin’s lymphoma: a population-based case-control study of men in Minnesota Arch Environ Health 55:326–329.11063407 Find this article online
32. Grant W, Steele G, Isiorho SA 1996. Spontaneous abortions possibly related to ingestion of nitrate-contaminated well water—LaGrange County, Indiana, 1991–1994 MMWR Morb Mortal Wkly Rep 45:569–572.9132576 Find this article online
33. Gulis G, Czompolyova M, Cerhan JR 2002. An ecologic study of nitrate in municipal drinking water and cancer incidence in Trnava District, Slovakia Environ Res 88:182–187.12051796 Find this article online
34. Gupta SK, Gupta RC, Gupta AB, Seth AK, Bassin JK, Gupta A 2000. Recurrent acute respiratory infections in areas with high nitrate concentrations in drinking water Environ Health Perspect 108:363–366.10753096 Find this article online
35. Gupta SK, Gupta RC, Seth AK, Gupta AB, Bassin JK, Gupta A 1999. Adaptation of cytochrome b5 reductase activity and methemoglobinemia in areas with a high nitrate concentration in drinking-water Bull WHO 77:749–753.10534899 Find this article online
36. Hanukoglu A, Danon PN 1996. Endogenous methemoglobinemia associated with diarrheal disease in infancy J Pediatr Gastroenterol Nutr 23:1–7.8811515 Find this article online
37. Haorah J, Zhou L, Wang X, Xu G, Mirvish SS 2001. Determination of total N-nitroso compounds and their precursors in frankfurters, fresh meat, dried salted fish, sauces, tobacco, and tobacco smoke particulates J Agric Food Chem 49:6068–6078.11743810 Find this article online
38. Hodgson RM, Wiessler M, Kleihues P 1980. Preferential methylation of target organ DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine Carcinogenesis 1:861–866.11219858 Find this article online
39. Hutson SS, Barber NL, Kenny JF, Linsey KS, Lumia DS, Maupin MA 2004. Estimated Use of Water in the United States in 2000. USGS Circular 1268. Denver, CO:U.S. Geological Survey
40. Jedrychowski W, Maugeri U, Bianchi I 1997. Environmental pollution in central and eastern European countries: a basis for cancer epidemiology Rev Environ Health 12:1–23.9128908 Find this article online
41. Kamiyama S, Ohshima H, Shimada A, Saito N, Bourgade MC, Ziegler P, et al. 1987. 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 84:479–502. Find this article online
42. Kimura H, Hokari R, Miura S, Shigematsu T, Hirokawa M, Akiba Y, et al. 1998. Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis Gut 42:180–187.9536941 Find this article online
43. Knobeloch L, Salna B, Hogan A, Postle J, Anderson H 2000. Blue babies and nitrate-contaminated well water Environ Health Perspect 108:675–678.10903623 Find this article online
44. Kostraba JN, Gay EC, Rewers M, Hamman RF 1992. Nitrate levels in community drinking waters and risk of IDDM, an ecologic analysis Diabetes Care 15:1505–1508.1468277 Find this article online
45. Lambert KF, Driscoll C 2003. Nitrogen Pollution: From the Sources to the Sea. Hanover, NH:Hubbard Brook Research Foundation
46. Lashner BA, Hanauer SB, Silverstein MD 1988. Optimal timing of colonoscopy to screen cancer in ulcerative colitis Ann Intern Med 108:274–278.3124681 Find this article online
47. Leaf CD, Wishnok JS, Tannenbaum SR 1989. L-Arginine is a precursor for nitrate biosynthesis in humans Biochem Biophys Res Commun 8:1032–1037. Find this article online
48. Lees Murdock DJ, Barnett YA, Barnett CR 2004. DNA damage and cytotoxicity in pancreatic beta-cells expressing human CYP2E1 Biochem Pharmacol 68:523–530.15242818 Find this article online
49. Le Marchand L, Donlon T, Seifried A, Wilkens LR 2002. Red meat intake, CYP2E1 genetic polymorphisms, and colorectal cancer risk Cancer Epidemiol Biomarkers Prev 11:1019–1024.12376502 Find this article online
50. Levallois P, Ayotte P, Van Maanen JM, Desrosiers T, Gingras S, Dallinga JW, et al. 2000. Excretion of volatile nitrosamines in a rural population in relation to food and drinking water consumption Food Chem Toxicol 38:1013–1019.11038239 Find this article online
51. Levine JJ, Pettei MJ, Valderrama E, Gold DM, Kessler BH, Tractman H 1998. Nitric oxide and inflammatory bowel disease: evidence for local intestinal production in children with active colonic disease J Pediatr Gastroenterol Nutr 26:34–38.9443117 Find this article online
52. L’hirondel J, L’hirondel J-L 2002. Nitrate and man: toxic, harmless, or beneficial? Wallingford, Oxfordshire, UK: CABI Publishing
53. Lijinsky W 1986. The significance of N-nitroso compounds as environmental carcinogens J Environ Sci Health C4:1–45. Find this article online
54. Longnecker MP, Daniels JL 2001. Environmental contaminants as etiologic factors for diabetes Environ Health Perspect 109:871–876.11744505 Find this article online
55. Lu SH, Ohshima H, Fu HM, Tian Y, Li FM, Blettner M, et al. 1986. 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 46:1485–1491.3943105 Find this article online
56. Lundberg JO, Herulf M, Olesen M, Bohr J, Tysk C, Wiklund NP, et al. 1997. Increased nitric oxide production in collagenous and lymphocytic colitis Eur J Clin Invest 27:869–871.9373768 Find this article online
57. Mirvish SS, Grandjean AC, Moller H, Fike S, Maynard T, Jones L, et al. 1992. N-nitrosoproline excretion by rural Nebraskans drinking water of varied nitrate content Cancer Epidemiol Biomarkers Prev 1:455–461.1302557 Find this article online
58. Mirvish SS, Haorah J, Zhou L, Hartman M, Morris CR, Clapper ML 2003. N-nitroso compounds in the gastrointestinal tract of rats and in the feces of mice with induced colitis or fed hot dogs or beef Carcinogenesis 24:595–603.12663523 Find this article online
59. Moller H, Landt J, Pedersen E, Jensen P, Autrup H, Jensen O 1989. Endogenous nitrosation in relation to nitrate exposure from drinking water and diet in a Danish rural population Cancer Res 49:3117–3121.2720669 Find this article online
60. Morales-Suarez-Varela MM, Llopis-Gonzalez A, Tejerizo-Perez ML 1995. Impact of nitrates in drinking water on cancer mortality in Valencia, Spain Eur J Epidemiol 11:15–21.7489769 Find this article online
61. Mueller BA, Newton K, Holly EA, Preston-Martin S 2001. Residential water source and the risk of childhood brain tumors Environ Health Perspect 109:551–556.11445506 Find this article online
62. National Academy of Sciences 1981. The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds. Washington, DC:National Academy of Sciences
63. National Research Council 1995. Nitrate and nitrite in drinking water. Washington DC:National Academy Press
64. Nolan BT, Hitt KJ, Ruddy BC 2002. Probability of nitrate contamination of recently recharged groundwaters in the conterminous United States Environ Sci Technol 36:2138–2145.12038822 Find this article online
65. Ohshima H, Bartsch H 1994. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis Mutat Res 305:253–264.7510036 Find this article online
66. Parslow RC, McKinney PA, Law GR, Staines A, Williams R, Bodansky HJ 1997. Incidence of childhood diabetes mellitus in Yorkshire, northern England, is associated with nitrate in drinking water: an ecologic analysis Diabetologia 40:550–556.9165223 Find this article online
67. Pomeranz A, Korzets Z, Vanunu D, Krystal H 2000. Elevated salt and nitrate levels in drinking water cause an increase of blood pressure in schoolchildren Kidney Blood Press Res 23:400–403.11070420 Find this article online
68. Roediger WE, Lawson MJ, Radcliffe BC 1990. Nitrite from inflammatory cells: a cancer risk factor in ulcerative colitis Dis Colon Rectum 33:1034–1036.2242698 Find this article online
69. Sanchez-Echaniz J, Benito-Fernandez J, Mintegui-Raso S 2001. Methemoglobinemia and the consumption of vegetables in infants Pediatrics 107:1024–1028.11331681 Find this article online
70. Sandor J, Kiss I, Farkas O, Ember I 2001. Association between gastric cancer mortality and nitrate content of drinking water: ecological study on small area inequalities Eur J Epidemiol 17:443–447.11855578 Find this article online
71. Schmitz JT 1961. Methemoglobinemia—a cause of abortions? Obstet Gynecol 17:413–414.13748137 Find this article online
72. Scragg RK, Dorsch MM, McMichael AJ, Baghurst PA 1982. Birth defects and household water supply. Epidemiological studies in the Mount Gambier region of South Australia Med J Aust 2:577–579.7162445 Find this article online
73. Shearer LA, Goldsmith JR, Young C, Kearns OA, Tamplin BR 1972. Methemoglobin levels in infants in an area with high nitrate water supply Am J Public Health 62:1174–1180.5068711 Find this article online
74. Shuval HI, Gruener N 1972. Epidemiological and toxicological aspects of nitrates and nitrites in the environment Am J Public Health 62:1045–1052.5046442 Find this article online
75. Simon C, Manzke H, Kay H, Mrowetz G 1964. On the incidence, pathogenesis and prevention of methemoglobinemia caused by nitrites [In German] Z Kinderheilkd 91:124–138. Find this article online
76. Spalding RF, Exner ME 1993. Occurrence of nitrate in ground-water—a review J Environ Qual 22:392–402. Find this article online
77. Spiegelhalter B, Elsenbrand G, Preussman R 1976. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds Food Cosmet Toxicol 14:545–548.1017769 Find this article online
78. Steindorf K, Schlehofer B, Becher H, Hornig G, Wahrendorf J 1994. Nitrate in drinking water. A case-control study on primary brain tumours with an embedded drinking water survey in Germany Int J Epidemiol 23:451–457.7960368 Find this article online
79. Stich HF, Dunn BP, Pignatelli B, Ohshima H, Bartsch H 1984. Dietary phenolics and betel nut extracts as modifiers of N-nitrosation in rat and man IARC Sci Publ 57:213–222.6533010 Find this article online
80. Super M, Heese HDV, MacKenzie D, Dempster WS, Du Plessis J, Ferreira JJ 1981. An epidemiological study of well-water nitrates in a group of South West African/Namibian infants Water Res 15:1265–1270. Find this article online
81. Suzuki T, Itoh S, Nakajima M, Hachiya N, Hara T 1999. Target organ and time-course in the mutagenicity of five carcinogens in MutaMouse: a summary report of the second collaborative study of the transgenic mouse mutation assay by JEMS/MMS Mutat Res 444:259–268.10521667 Find this article online
82. Tricker AR 1997. N-nitroso compounds and man: sources of exposure, endogenous formation and occurrence in body fluids Eur J Cancer Prev 6:226–268.9306073 Find this article online
83. Tricker AR, Mostafa MH, Spiegelhaler B, Pruessmann R 1989. Urinary excretion of nitrate, nitrate and N-nitroso compounds in Schistosomiasis and bilharzia bladder cancer patients Carcinogenesis 10:547–552.2924399 Find this article online
84. U.S. EPA 1991. Integrated Risk Information System (IRIS): Nitrate (CASRN 14797-55-8). Washington, DC:U.S. Environmental Protection Agency. Available: http://www.epa.gov/iris/subst/0076.htm [accessed 31 May 2005]
85. van Loon AJ, Botterweck AA, Goldbohm RA, Brants HA, van Klaveren JD, van den Brandt PA 1998. Intake of nitrate and nitrite and the risk of gastric cancer: a prospective cohort study Br J Cancer 78:129–135.9662263 Find this article online
86. van Maanen JM, Pachen DM, Dallinga JW, Kleinjans JC 1998. Formation of nitrosamines during consumption of nitrate and amine-rich foods, and the influence of mouthwashes Cancer Detect Prev 22:204–212.9618041 Find this article online
87. van Maanen JM, van Dijk A, Mulder K, de Baets MH, Menheere PC, van der Heide D, et al. 1994. Consumption of drinking water with high nitrate levels causes hypertrophy of the thyroid Toxicol Lett 72:365–374.8202954 Find this article online
88. van Maanen JM, Welle IJ, Hageman G, Dallinga GW, Mertens PL, Kleinjans JC 1996. Nitrate contamination of drinking water: relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of N-nitrosamines Environ Health Perspect 104:522–528.8743440 Find this article online
89. van Maanen JMS, Albering HJ, de Kok TMCM, van Breda SGJ, Curfs DMJ, Vermeer ITM, et al. 2000. Does the risk of childhood diabetes mellitus require revision of the guideline values for nitrate in drinking water? Environ Health Perspect 108:457–461.10811574 Find this article online
90. Vermeer I, Pachen DM, Dallinga JW, Kleinjans JC, van Maanen JM 1998. Volatile N-nitrosamine formation after intake of nitrate at the ADI level in combination with an amine-rich diet Environ Health Perspect 106:459–463.9681972 Find this article online
91. Walton G 1951. Survey of literature relating to infant methemoglobinemia due to nitrate-contaminated water Am J Public Health 41:986–996.14847023 Find this article online
92. Ward MH, Cantor KP, Cerhan J, Lynch CF, Hartge P 2004. Drinking water nitrate and cancer: results from recent studies in the Midwestern United States [Abstract] Epidemiology 15:S214. Find this article online
93. Ward MH, Cantor KP, Riley D, Merkle S, Lynch CF 2003. Nitrate in public water supplies and risk of bladder cancer Epidemiology 14:183–190.12606884 Find this article online
94. Ward MH, Mark SD, Cantor KP, Weisenburger DD, Correa-Villasenor A, Zahm SH 1996. Drinking water nitrate and the risk of non-Hodgkin’s lymphoma Epidemiology 7:465–471.8862975 Find this article online
95. Wennmalm A, Benthin G, Edlund A, Jungersten L, Kieler-Jensen N, Lundlen S, et al. 1993. Metabolism and excretion of nitric oxide in humans: an experimental and clinical study Circ Res 73:1121–1127.8222083 Find this article online
96. Weyer PJ, Cerhan JR, Kross BC, Hallberg GR, Kantamneni J, Breuer G, et al. 2001. Municipal drinking water nitrate level and cancer risk in older women: the Iowa Women’s Health Study Epidemiology 12:327–338.11338313 Find this article online
97. WHO 2004a. Nitrates and Nitrites in Drinking-Water. WHO/SDE/WSH/04.08/56. Rolling revision of the WHO guidelines for drinking-water quality. Draft for review and comments. Geneva:World Health Organization. Available: http://www.who.int/water_sanitation_heal​th/dwq/chemicals/en/nitratesfull.pdf [accessed 31 May 2005]
98. WHO 2004b. Guidelines for Drinking Water Quality. 3rd Ed, Vol. 1, Recommendations. Geneva:World Health Organization
99. Zeman CL, Kross B, Vlad M 2002. A nested case-control study of methemoglobinemia risk factors in children of Transylvania, Romania Environ Health Perspect 110:817–822.12153765 Find this article online