NO and NOS are known to be involved in cancer-related events (angiogen- esis, apoptosis, cell cycle, invasion, and metastasis) and are linked to increased oxidative stress and DNA damage (Ying and Hofseth, 2007). There is evi- dence that dietary patterns, foods, nutrients, and other dietary constituents are closely associated with the risk for several types of cancer. And while it is not yet possible to provide quantitative estimates of the overall risks, it has been estimated that up to 35% of cancer deaths may be related to dietary factors (Doll and Petro, 1981). Nitrate exposure through forma- tion of N-nitrosamines has been associated with negative health effects due to increased risk for certain cancers, primarily gastric and colon cancers.
In 1976, Spiegelhalder in Germany and Tannenbaum in the USA showed that nitrate could theoretically be transformed into N-nitrosamines by the reaction of salivary nitrite with secondary amines in the diet (Spiegelhalder et al., 1976; Tannenbaum et al., 1976). Since then, when the formation of potentially carcinogenic N-nitrosamines from the reaction of HNO2 with secondary amines was recognized, the use of nitrate and nitrite salts as food preservatives has come under intense scrutiny. It has been known since 1956 that N-nitrosamines could cause hepatic tumors in laboratory animals by reacting with nucleic acids (Magee and Barnes, 1956). This reached a crisis in 1979 when Newberne published a report that dietary nitrite caused lymphomas in rats although not thought to occur through the formation of N-nitrosamines (Newberne, 1979). Nitrite has been used for centuries to protect from food-borne illnesses. There is no other effective way to kill botulinum spores which are resistant even to boiling (Pierson and Smoot, 1982). Although further analysis of Newberne’s results and further work carried out by the Food and Drug Administration failed to confirm that nitrite causes cancer, this initial report along with the clear association with N-nitrosamines and cancer created the public perception that nitrite and nitrate are harmful and should be avoided. Public policy also intervened during this time to quantify and limit exposure to nitrite and nitrate. A large number of studies were undertaken, many of which purported to show a positive link between nitrate intake and cancer. Experimental evidence does indicate that co-administering a low-molecular-weight amine with nitrite can cause N-nitrosamine formation (Sen et al., 1969). Nitrosamines have been shown to cause a wide range of tumors in more than 40 animal spe- cies and may be specifically involved in the etiology of gastric cancer and esophageal cancer (Tricker and Preussmann, 1991) although so far, there is
no conclusive epidemiologic evidence that these compounds are related to cancer risk in humans.
Numerous case–control studies have been conducted worldwide to determine if there is a link between gastric cancer and nitrate intake (Blot et al., 1999; Eichholzer and Gutzwiller, 1998; Moller, 1995). It is well known that elevated dietary nitrate intake leads to elevated salivary nitrate levels and, after reduction by oral bacteria, higher levels of ingested nitrite. Studies in Canada, Italy, Sweden and Germany involving thousands of study subjects have failed to show an association or have demonstrated an inverse association between estimated nitrate intake and gastric cancer, perhaps because much of the nitrate was from vegetables (Moller, 1995).
Occupational exposure to very high levels of nitrate occurs in nitrate fer- tilizer workers, who have elevated body burdens of nitrate and elevated salivary nitrate and nitrite levels, but no increase in gastric cancers has been observed (Moller, 1995). Case–control studies attempting to link nitrate and nitrite consumption to brain, esophageal, and nasopharyngeal cancers have also been inconclusive (Eichholzer and Gutzwiller, 1998). In other studies published over two decades, the relationship between the consumption of cured meats during pregnancy and the risk of brain and other tumors in offspring was examined (Blot et al., 1999). In this review of 14 epidemio- logical studies, 13 of which were case–control studies, Blot et al. (1999) could not conclude that there was a relationship between cured meat con- sumption during pregnancy and brain or any other cancers. It may be that in the limited number of epidemiological studies linking nitrate, nitrite or cured meat to a specific cancer site, other as-yet uncharacterized dietary or environmental factors may be involved. Most if not all of the epidemio- logical studies showing a relationship between nitrate exposure and cancer are very weak, and in 2003, the Joint FAO/WHO Expert Committee on Food Additives pronounced “Overall, the epidemiological studies showed no consistently increased risk for cancer with increasing consumption of nitrate. These data, combined with the results of the epidemiological studies considered by the Committee at its 44th meeting, do not provide evidence that nitrate is carcinogenic to humans” (Speijers and Brandt, 2003). Subse- quent epidemiological studies have similarly failed to show a convincing link between nitrate intake and cancer. In fact, many experts suggest that any epidemiological study with a relative risk index of <2.0 should not be used for public policy recommendations (Anderson, 1994; Rosenberg, 1994) due to chance, statistical bias or effects of confounding factors that are sometimes not evident. Many epidemiologic studies have shown that
populations that eat diets high in vegetables and fruits and low in animal fat, meat, and/or calories have reduced risk of some of the most common can- cers. Coincidently, fruits and vegetables are enriched with nitrite and nitrate from the soil. The presence of numerous classes of antioxidants is generally accepted as the explanation for the preventive effect of vegetables con- sumption, which may prevent oxidative stress and nitrosative DNA damage (Nishino et al., 2005). Despite this, it is entirely possible that certain sub- groups of patients may have a higher risk of cancer when exposed to high levels of dietary nitrate. These at-risk populations will have to be identified.
It is possible that future research will show that there are some groups, in whom it may be advisable to reduce nitrate exposure similar to groups that should avoid coffee or other otherwise safe products.
In view of the complex context and multiple cofactors of nitrate in diet-related carcinogenesis, it is not surprising that epidemiological studies into the relation between nitrate in drinking water and cancers (or other health effects) often provide weak associations, and both positive and nega- tive (Ward et al., 2005). Ward et al. (2005) conclude that “the few stud- ies that have evaluated intake of nitrosation precursors or inhibitors have observed elevated risks for colon cancer and neural tube effects associated with drinking-water nitrate concentrations below the regulatory limit.” For example, De Roos et al. (2003) found that the odds ratio for colon cancer (which is equivalent to the relative incidence) almost doubled (95% CI) for the subgroup with above-median meat intake and exposed for more than 10 years to nitrate concentrations in drinking water exceeding 5 mg N/L (22 mg NO3/L, which is half the legal US limit) as compared to the refer- ence group that was not exposed (Fig. 3.4). This study did not find an asso- ciation between colon cancer and nitrate for the total population. However, the European Food Safety Authority (Authority, 2008) concluded from a review of recent epidemiological studies, “these were mostly studies with a weak study design and limited strength of evidence; other case–control stud- ies and cohort studies (which provide stronger evidence) find no increased risk with increasing nitrate intake after multivariate adjustment.”
Many, if not most, of the early studies on nitrate and cancer were con- ducted before it was realized that nitrate is actually produced endogenously in the body. Endogenous NO activity leads to an increase in blood and tissue nitrate. In fact, it has been estimated that at least half of human nitrate exposure comes from endogenous production of NO and the other half from what we eat. A report from the National Research Council (The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds, NRC, 1981)
(Pennington, 1998) estimated based on food-consumption tables that the average total nitrite and nitrate intake in the United States was 0.77 mg and 76 mg, respectively. If we assume our body (70 kg) produces 1.68 mmol NO per day (based on 1 àmol/kg/h NO production). An average daily
Figure 3.4 Increase of colon cancer incidence by drinking water nitrate in Iowa pub- lic water supply (and 95% confidence intervals) for subgroups with above and below median dietary and medical risk factors, and with 1–10 years of exposure and more than 10 years of exposure to drinking water exceeding 5 mg NO3-N/L, relative to the subgroup with no exposure (van Grinsven et al., 2010, inferred from De Roos et al., 2003).
For color version of this figure, the reader is referred to the online version of this book.
intake of 0.77 mg of nitrite would equate to 11.1 àmol per day and 76 mg nitrate would equate to 894 àmol per day or roughly 1 mmol nitrite and nitrate per day from diet. This almost matches what our body makes from NO if we assume most of the NO goes to stepwise oxidation to nitrite and nitrate. The dietary exposure of nitrate is even higher in vegetarians and in the populations in the Mediterranean region. Furthermore, physical activity enhances NO output and leads to a concomitant increase in nitrate.
Therefore, our steady-state level of nitrate is derived almost 50% from diet and lifestyle. We now know that increasing endogenous exposure of nitrate is a natural and adaptive physiological response. In comparison to humans living at sea level, high-altitude residents of Tibet (4200 m above seal level) have >10-fold-higher circulating concentrations of bioactive NO, including nitrite and nitrate (Erzurum et al., 2007). There does not appear to be any adverse consequences to this population as a result of their increased nitrate exposure from endogenous synthesis of NO despite decades of exposure to these high concentrations of nitrate.
Early reports indicated dietary nitrate may affect iodine metabolism and thyroid function (Bloomfield, et al., 1961). More recent data indicate that relatively low doses exceeding the ADI for nitrate by three times have no effect on thyroid function (Hunault et al., 2007). However, emerging epi- demiological data suggest that dietary nitrate exposure may be associated with thyroid cancer (Kilfoy et al., 2011). Additional research is needed to determine the role of nitrate and thyroid function and cancer.
In 2006, a review of ingested nitrate and nitrite carcinogenicity was con- ducted by an expert working group convened by the International Agency for Research on Cancer (IARC), a United Nations/WHO headquartered in Lyon, France. The agency is involved in both epidemiological and labo- ratory research and disseminates scientific information through publica- tions, meetings, courses, and fellowships. The IARC working group, whose report is not yet publicly available except in summary form IARC (2010) (http://monographs.iarc.fr/ENG/Monographs/vol94/mono94.pdf), clas- sified nitrate and nitrite as a probable carcinogenic agent to humans:
“Ingested nitrate or nitrite under conditions that result in endogenous nitrosation is probably carcinogenic to humans (Group 2A).”
This category is used when there is limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals.
In some cases, an agent may be classified in this category when there is inadequate evidence of carcinogenicity in humans and sufficient evidence of
carcinogenicity in experimental animals and strong evidence that the car- cinogenesis is mediated by a mechanism that also operates in humans.
Exceptionally, an agent may be classified in this category solely on the basis of limited evidence of carcinogenicity in humans. An agent may be assigned to this category if it clearly belongs, based on mechanistic considerations, to a class of agents for which one or more members have been classified in Group 1 or Group 2A.
The expert group found that nitrate/nitrite was only weakly associated with human stomach cancer and no other cancers. In simple terms, this overall evaluation means that the ingestion of food and water that contain nitrate or nitrite (e.g. spinach and other green leafy vegetables, root veg- etables, bread, beer, cured meats), in combination with amines and amides commonly found in food, can react in the stomach to form N-nitrosamines and N-nitrosamides, which are known animal carcinogens already classi- fied by IARC. However, the classification of nitrite and nitrate as Group 2A appears unwarranted and unjustified since there is no evidence based on new data demonstrating no association between nitrite and nitrate and gastric or esophageal cancer (Cross et al., 2010) combined with limited evidence in experimental animals.