Association between Dietary Nitrate, Nitrite Intake, and Site-Specific Cancer Risk: A Systematic Review and Meta-Analysis

Background: People consume nitrates, nitrites, nitrosamines, and NOCs compounds primarily through processed food. Many studies have yielded inconclusive results regarding the association between cancer and dietary intakes of nitrates and nitrites. This study aimed to quantify these associations across the reported literature thus far. Methods: We performed a systematic review following PRISMA and MOOSE guidelines. A literature search was performed using Web of Science, Embase, PubMed, the Cochrane library, and google scholar up to January 2020. STATA version 12.0 was used to conduct meta-regression and a two-stage meta-analysis. Results: A total of 41 articles with 13 different cancer sites were used for analysis. Of these 13 cancer types/sites, meta-regression analysis showed that bladder and stomach cancer risk was greater, and that pancreatic cancer risk was lower with increasing nitrite intakes. Kidney and bladder cancer risk were both lower with increasing nitrate intakes. When comparing highest to lowest (reference) categories of intake, meta-analysis of studies showed that high nitrate intake was associated with an increased risk of thyroid cancer (OR = 1.40, 95% CI: 1.02, 1.77). When pooling all intake categories and comparing against the lowest (reference) category, higher nitrite intake was associated with an increased risk of glioma (OR = 1.12, 95% CI: 1.03, 1.22). No other associations between cancer risk and dietary intakes of nitrates or nitrites were observed. Conclusion: This study showed varied associations between site-specific cancer risks and dietary intakes of nitrate and nitrite. Glioma, bladder, and stomach cancer risks were higher and pancreatic cancer risk was lower with higher nitrite intakes, and thyroid cancer risk was higher and kidney cancer risk lower with higher nitrate intakes. These data suggest type- and site-specific effects of cancer risk, including protective effects, from dietary intakes of nitrate and nitrite.


Introduction
Cancer is a leading cause of death worldwide, accounting for nearly 10.0 million (approximately one in six) deaths, in 2020 [1]. The global cancer burden is expected to be 28.4 million cases in 2040, a 47% rise from 2020 [1]. With this growing global burden, more evidence-based practice is needed in the identification and management of risk factors for cancer development. Although the causes of cancer are not completely understood, numerous factors are known to increase risk, including non-modifiable factors (e.g., gender,

Discussion
This systematic literature review and meta-analysis aimed to quantify the associations between cancer risk and dose of dietary nitrate and nitrite reported in the literature to date. Using 41 eligible articles, we conducted meta-analyses using two different approaches to compare the risk of 13 different site-specific cancers across different categories of dietary intake. Moreover, we conducted a meta-regression analysis to examine associations between site-specific cancer risk and dosage of dietary nitrates and nitrites.
Firstly, when comparing highest to lowest (reference) categories of intake, metaanalysis showed that high nitrate intake was associated with an increased risk of thyroid cancer (OR = 1.40, 95% CI: 1.02, 1.77). When pooling all intake categories and comparing against the lowest (reference) category, higher nitrite intake was associated with an increased risk of glioma (OR = 1.12, 95% CI: 1.03, 1.22).
Meta-regression analysis showed that bladder and stomach cancer risk was greater, and that pancreatic cancer risk was lower, with increasing nitrite intakes. Kidney and bladder cancer risk were both lower with increasing nitrate intakes. No other associations between cancer risk and dietary intakes of nitrates or nitrites were observed. These data suggest type-and site-specific effects on cancer risk, including protective effects, from dietary intakes of nitrate and nitrite.
These findings from a meta-analysis of the literature are an important contribution, as individual studies on their own have reported seemingly inconsistent findings. Some studies have shown positive, and others, negative associations with cancer risk at different intakes of dietary nitrate and nitrite. For example, Kilfoya et al. [51] reported an association between ovarian cancer and a daily nitrate intake of 175.4 mg/day, HR: 1.31 (95% CI: 1.01, 1.68) in a 10-year prospective cohort study of women (aged 50-71 years), with a total of 709 incident epithelial ovarian cancer cases. This same study did not show any association between ovarian cancer and total nitrate intake, yet there was a relationship between a nitrate intake of 0.33 mg/1000 kcal from animal sources HR: 1.34 (95% CI: 1.05, 1.69). In contrast, Inoue-Choi et al., [5] did not show any association with the same range of nitrate daily intake 165.48-209.2 mg/day, HR: 0.85 (95% CI: 0.56, 1.27) in a similar cohort study of women aged 55-69 years. This is a good comparison because these two studies have almost the same daily nitrate intake and the same demographic characteristics, which is sometimes difficult to find. Three studies on breast cancer did not show any associations [12,52,53]. More research is needed to study the association between nitrate and nitrite intake and breast cancer from both food and water, especially since it is the second leading cause of cancer death in 92% of women. Non-Hodgkin's lymphoma studies showed an association between daily nitrite intake and the disease [25,56,60]. Most studies had daily intake ranges of 0-2 mg/day; one study did not report the daily intake [25]. There was no association between daily nitrate intake and this cancer. Stomach cancer was the most studied cancer among the articles retrieved for this systematic review, yet only three of these studies showed any relationship with dietary nitrite intake. This study's meta-regression showed an association between dietary nitrite exposure and stomach cancer. Daily nitrate intake was not associated with stomach cancer in the meta-regression analysis. As for other cancers (cancer of the colon, rectum, esophagus, pancreas, kidney, thyroid, and glioma), one or two studies showed positive associations with dietary nitrate and nitrite intake.
Many studies have shown that a long period of exposure/daily dietary intake that contains nitrate, nitrite, and NOC compounds can lead to specific health issues, but these results are still contradictory. Keszei, et al. (2013) [69] conducted a cohort study with 16.3 years of follow-up in the Netherlands, from 1986 to 2002 for men and women aged 55-69 years. In this study, esophageal squamous cell carcinoma (ESCC) risk was associated with nitrite intake (HR for 0.1-mg/day increase: 1.19; 95% CI: 1.05, 1.36; p-trend = 0.06). Positive associations were observed between N-nitrosodimethylamine intake and esophageal squamous cell carcinoma (ESCC) risk (HR for 0.1 micro gram/day increase in intake: 1.15; 95% CI: 1.05, 1.25; p-trend = 0.01 based on tertiles of intake) and gastric non-cardia adenocarcinoma (GNCA) risk (1.06; 95% CI: 1.01, 1.10; p-trend = 0.09) in men. Meanwhile Cross et al., 2011, [53] conducted a cohort study with 10 years of follow-up in the USA, from 1995 to 2006 for men and women aged 50-71 years, which showed nitrate and nitrite were not associated with esophageal or gastric cancer. Some case-control studies and ecological studies have yielded inconclusive results about different types of cancers.
Individually, most of the research articles included in this systematic review did not find any association between nitrate and nitrite and any type of cancer in humans. However, when analyzed together, greater exposure to dietary nitrate and nitrite increased the risk of getting some cancers (glioma, bladder, stomach, and thyroid) and decreased the risk of getting others (pancreatic and kidney). Most of these cancers can be considered cancers of the digestive system. The risk of these types of cancers have been shown to be modified by other dietary and lifestyle factors. For example, some studies have shown an inverse association of vegetable and fruit intake with these cancers' risk [78]. Other studies have shown that people who take in high vitamin C, high vitamin E, low red meat (or any type of meat), and folate while being exposed to nitrate or nitrite at the same time had a lower risk of having cancer, than those who did not [64,79]. However, not all studies showed these protective effects. Zeegers et al., 2006, [65] showed that vitamin C (p = 0.63) and vitamin E (p = 0.62), did not appear to be significant effect modifiers in the association between nitrate exposure from food and bladder cancer risk. Catsburg et al., (2014) [38] showed that among individuals with high nitrate intake, a positive association between high (i.e., above the median) heme intake and risk of bladder cancer was observed (highest category vs. lowest category OR = 1.76; 95% CI = 1.21-2.55; p trend = 0.007). Some studies showed no association at all [33,67,80].
Notwithstanding the null findings of some studies, it is widely accepted that it is important that people consume diets high in fresh fruits and vegetables that contain a lot of vitamins and essential minerals and reduce meat, fatty food, and processed food intake to improve their health. This may be important in modifying any harmful effects of dietary nitrates and nitrites on particularly susceptible tissues in the digestive system and elsewhere in the body. Cases of cancer are considered to be linked to nutritional factors. Scientific evidence suggests that food/diet is most convincingly linked to cancer of the lung, stomach, rectum, colon, pharynx, nasopharynx, esophagus, and mouth [81][82][83]. Filtration and purification of drinking water from both private and public sources before consumption is extremely important because several studies have shown that the consumption of nitrate and nitrite from drinking water, even in a very small amount, over a long period can lead to cancer (a chronic disease) and other health issues [31,84,85].
Most of the studies included in this systematic review were conducted in Europe and the U.S., and very few or no studies from South America, Africa, Australia, and Asia were retrieved. Many studies and awareness of early screening for different types of cancers are needed in South America, Africa, Australia, and Asia to better understand this issue on a larger scale with different demographics. A proper and comprehensive assessment of nitrate and nitrite from dietary intake, including inhibitors of endogenous nitrosation and intakes of antioxidants, are needed in future studies. Many studies lacked information about study participants' water consumption and dietary intake that contain nitrate and nitrite simultaneously, which may be essential to better analyze the links between dietary intakes and cancers. Future studies should also pay close attention to the different duration or lengths (years) of food intake with nitrite and nitrate, especially to understand the effects of exposure. There is still no precise standard maximum contaminant level for nitrate and nitrite in food to protect people from non-communicable diseases like cancer. This might be because noncommunicable diseases, such as cancer, can take a long time to occur and the casualty has not yet been fully established.

Limitations
Using two different statistical analyses for each cancer site is the strength of this research. It can help to better understand the robustness of the associations. However, the study's limitations are as follows; first, very few articles were available for each type of cancer, some with three or fewer studies, and so the results for this analysis should be treated with caution. More detailed, well-designed studies with accurate and precise information about study participants' food intake and other lifestyle factors may be essential to more accurately estimate associated risk. Secondly, this research was unable to adjust for potential confounders or examine effect modification (e.g., of dietary vitamin C intake) due to lack of available data in the included studies. Some studies included vitamin C, D, and E, and folate and red/processed meat intake; however, most did not.
We recommend that such research should include dietary vitamin C, D, and E, intake, as well as folate intake, polyphenols, red/processed meat, heme iron intake, and other nutrients/minerals and compounds (especially the dosage) from food and drinking water that could affect nitrosation in the body, to enable more precise estimation of risks and more sophisticated analyses.
Third, there was a wide range of nitrate and nitrite intake values from different studies, which resulted in different ranges across the analyses performed in this meta-analysis. For example, dietary nitrite ranged from 0 to 2.4 mg/day in the analysis against bladder cancer risk and from 0 to 22 mg/day in the analysis against stomach cancer risk. This variation in range in the independent variable across different analyses could be partly responsible for the variation in association with the dependent variables (the different site-specific cancer risks) observed in this study. A lack of standardized units for reporting dietary intakes of nitrates and nitrites in the literature made it necessary to convert the data to a common unit for meta-regression. Whilst this is not a major limitation, it has the potential to introduce error. Moreover, a lack of standardized units might pose a problem to efforts to implement a limit or precise standard maximum contaminant level to protect people from health risks of having a type of cancer. Lastly, many cohort and case-control studies should be done in different parts of the world to understand this topic better, especially noting other confounding factors and nutrient intake.

Conclusions
This study showed varied associations between site-specific cancer risks and dietary intakes of nitrate and nitrite. Glioma, bladder, and stomach cancer risks were higher, but pancreatic cancer risk was lower with higher nitrite intakes. Thyroid cancer risk was higher, but kidney and bladder cancer risks were lower with higher nitrate intakes. These data suggest type-and site-specific effects of cancer risk, including protective effects, from dietary intakes of nitrate and nitrite.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article. The data set used/analyzed are available from the corresponding author on request.