Is my Salami Giving me Cancer? Myths & Misinformation

There is an odd but enduring concept that dietary nitrate or nitrite can be a positive driver of cancer via the formation of nitrosamines. This misconception arose from overzealous interpretation errors and & incorrect extrapolation that emerged some decades ago and has stuck in the mindset, sadly. The misinterpretation arose before we appreciated the implications of endogenous production of nitric oxide and reactive nitrogen oxides. The purpose of this article is to help explain why dietary nitrate or nitrite, for example in your salami or hot dogs DO NOT cause cancer. 

Sources of Nitrate (NO3-) and Nitrite (NO2-) in the Body

There are three primary sources: food, water and endogenous production based on the conversion of arginine to nitric oxide. Let’s address their importance and some history.

Food accounts for the majority of ingested nitrate, which in turn is converted to nitrite by salivary bacteria utilizing an enzyme called nitrate reductase enzyme (1). It is present in our food predominantly in leafy green vegetables as their concentrate nitrate present in the soil as fertilizer. Nitrate rich dietary supplements, for example beet or spinach extracts, are now available and are used for cardiovascular health optimization and improved sports performance. It is also present in preserved meat (primarily as nitrite), although the levels of nitrate are considerably lower than vegetables.

Water, particularly well water, can be a source of nitrate. Early on, water-based nitrate was thought to be responsible for methemoglonemia in children via actions of cyanobacteria that readily convert the nitrate to nitrite (2). However, we now appreciate that the problem arises from an overly robust immune response to the microbial contamination of the well water not the nitrate per se. The immune response is characterized by excessive production of nitric oxide from endogenous sources, specifically a hitherto dormant enzyme that produces nitric oxide in vast amounts to defend against microbial invaders. The rules governing the amounts of nitrate/nitrite in our water were developed before we were aware of these endogenous sources and as a result, they may not always make sense. For example the amount of nitrite in saliva substantially exceeds the upper limit for water as set by the EPA. Does that mean we need to ban saliva?

In terms of the endogenous production there are 3 enzymes that make nitric oxide (NO) from L-arginine: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The latter is a dormant enzyme that is most commonly associated with activation of the immune system and inflammation. This isoform, iNOS, produces greater amounts of NO than either of the constitutive isoforms (nNOS, eNOS) because the iNOS form is constantly in the activate state. As long as there is substrate (L-arginine) it will make nitric oxide. This is the reason why the substrate, L-arginine, is classified as a semi-essential amino acid. It is only essential in states of immune activation where it is consumed by iNOS on such a scale to make massive amounts of NO that dietary amounts need to be in surplus to maintain its levels (3). Otherwise, the conversion of citrulline to arginine is adequate to maintain adequate arginine levels over the long term. 

There is another endogenous source of nitric oxide, and that is the recycling of nitrate to nitrite and then to NO. This occurs on a “need be” basis, catalyzed by acidic conditions, certain enzymes linked to ischemic states. Once NO is formed it is rapidly oxidized back to nitrate and nitrite. 

Because iNOS produces large amounts of NO, this is the isoform linked to the generation of reactive oxygen species that can lead to nitrosamine formation and cellular damage. These alternative reactive nitrogen species are the sources of complications not nitrate/nitrite.

Misconceptions about Sources as Drivers of Cancer

The original misconception on sources of nitrate/nitrite as it relates to cancer was via water. It was felt that water with high levels of nitrate could provoke a higher risk for colon cancer via the formation of nitrosamines. 

Several observations challenge this concept. Firstly, if this was true then the diets that are the richest in nitrate (plant-based and specifically leafy green, beetroot etc.) would have the highest rates of cancer. Logically, the more nitrate the more cancer. However, the epidemiology clearly shows the opposite. The more nitrate there is in the diet the LOWER the cancer rates (4-8). 

If simply ingesting nitrate was the problem, then the cancer rates would be higher, not lower. In short, the epidemiology fails to support a link to cancer, and actually suggests that a nitrate rich diet may offer a degree of protection as it does in cardiovascular health (9, 10).

There is a well-described nitrogen cycle within the body. Nitrate in plasma is actively pumped into the saliva, where it is converted into nitrite by salivary bacteria, swallowed and then absorbed from the GI tract. That nitrite is converted to nitric oxide as needed and then oxidized back to nitrate, and the cycle repeats.  

The purpose of this cycle is to maintain a constant reservoir of nitrate/nitrite from which the body can form nitric oxide for function and performance. The body is not wasteful in its design. We need a diet rich in nitrate in order to replenish the levels lost via urine and feces. It is like filling the bathtub without putting a plug in it. We need those dietary sources to be high for optimal levels. Retention systems within the body, like the pump from plasma into saliva, are not created to produce carcinogens, they are created to retain valuable resources.

Acid and Nitrate

Given that our saliva has a high nitrite concentration and when swallowed will interact with gastric acid it poses the risk of forming nitrosamines. Under lab bench conditions this can occur. If that is all that is needed to form dangerous levels of nitrosamines then our saliva would be classed as a source of cancer. Given that the rates of stomach cancer remain low and declining, together with the continued generation of nitrite-rich saliva, one must conclude that this explanation is inherently flawed. Hence, we must find an alternative explanation

Infection and Stomach Cancer

Research has now identified the pathogen microbe, Helicobacter pylori, as being responsible for gastric cancer. The immune system struggles to kill the bacteria as it hides out in stomach mucus in its own cloud of ammonia to neutralize gastric acid. The chemical bombs made by the immune response to the continued presence of H. pylori are continually formed in the gastric mucosa, include reactive nitrogen species that cause cellular damage and can form nitrosamines. In other words, gastric cancer is a result of an inflammatory insult from sustained immune activation not because I ate some salami the other day.

Another consideration is the concept of dose, which is often neglected. Within the context of the chemistry of reactive nitrogen species this is vital. For example, with the original contaminated water theory, we failed to identify the true problem - the body’s immune response to the microbes present in the water.

Why is this dose distinction critical? It is important because the amount of reactive nitrogen species generated by the immune/inflammatory system far exceeds the amount of nitrate that is present in the water. It also exceeds the level of nitrite in saliva for that matter. This production is based on the expression of the normally dormant isoform, iNOS, which generates large amounts of NO for as long as arginine is available.

Another way to look at it is to look at a cardiovascular system response to systemic infection – septic shock. In this scenario excess nitric oxide is produced by iNOS, as a result vascular resistance and blood pressure plummet, along with organ perfusion and function. If you trap the excess production of NO, blood pressure is corrected, and organ function is restored (11). 

While ingested dietary nitrate is able to lower blood pressure, the effect is small by comparison. Ingested nitrate can help manage hypertension but it never causes hypotension. In essence, the capacity to form NO and reactive nitrogen species with ingested nitrate is limited when compared to robust amounts generated from an activated immune system.

Generation of Nitrosamines from Acidified Nitrite

It is worth emphasizing but nitrite cannot form a nitrosamine directly. However, in the presence of acid, as in the stomach, nitrite can result in the formation of HNO2, which in turn can be converted to other reactive nitrogen species like the brown gases in smog (NO2 and N2O3), which are potent nitrosating agents. In the case of N2O3, it can break down to nitrite and nitrosonium (the nitrosating species).

HNO2 -----> NO+ + OH-

N2O3 ------> NO+ + NO2-

To further that concept, it is possible that when amines are ingested with nitrite and the stomach has a low pH, you can detect the formation of nitrosamines (23). Often the science stops here as Proof of Concept. But several questions are not addressed:

1. Firstly, in this highly manipulated and simplified scenario are the amounts generated meaningful? It is not sufficient to define their mere presence, as cancer represents a multisystem failure.
2. When the nitrate is ingested in its normal matrix – food – these reactions are greatly reduced, further lowering the generated dose. Antioxidants in particular are effective in limiting the formation of nitrosamines from acidified nitrite.

3. Further, if this is the mechanism of action (generation from stomach acid) does the generated nitrosamine reach a target organ in sufficient amounts to provoke a cancerous state (as opposed just stomach cancer from local production).

4. Given that the levels of nitrite in saliva usually exceed the limits of ADI in water, why then is not saliva simply driving an epidemic of gastric cancer?

Chronic Infection & Inflammation and Cancer

There are clear examples where an infection is associated with cancer. Hepatitis C provokes liver cancer through similar mechanisms (12), as does the bacteria, Helicobacter pylori), which resides in the mucus layer lining the stomach lumen, and is the only bacteria classed as a carcinogen by the WHO. (13). Try as it may, the immune response fails to clear these infective agents, but the reactive nitrogen species that are generated, can damage the local tissue through “friendly fire”. Cancer can be one of the results of this sustained burden.

My research team some 20+ years ago followed the anatomical localization of the chemical finger prints for nitration/nitrosation chemistry as well as cell death, in subjects carrying the burden H. pylori. They noted the source of this chemistry was the endogenous production of NO via iNOS. It was not linked to acid related gastric luminal production. In other words, this chemistry was linked to a heightened immune response to the H. pylori infection and not ingested nitrate or nitrite. Further, when supplemented with vitamin C these chemical fingerprints were suppressed (14), and the cancerous process placed in reverse (15).

Those results in gastric cancer identify that the nitrogen oxides may well be linked to gastric cancer, but the source of the problem lay in the immune response, not the generation of nitrogen oxides in the lumen of the stomach through interactions with acid. Nevertheless, the mythology of dietary nitrate persists.

While the International Agency for Research on Cancer (IARC) focuses on gastric cancer, many researchers have explored a potential role for dietary nitrate/nitrite in cancer at other systemic sites. One study in ovarian cancer (16) failed to find an association with total ingested nitrate/nitrite. When they broke down the dietary sources, they failed to find an association between plant-derived nitrite and that from processed meats but there was a general association with meat consumption and ovarian cancer. Meat-based diets have a well described higher risk for cancer, but it is not readily attributable to nitrite here, as the richest sources (plants and processed meats) failed to show an association. This is a clear case of investigator bias, looking at any possible way to force the data into a specific conclusion. 

How do you form a Nitrosamine?

Let’s be clear from the outset neither nitrate nor nitrite can directly form a nitrosamine from a secondary or tertiary amine. What is required is that both nitrate and nitrite be converted to other reactive nitrogen species, which then act as the nitrosating agents. This prerequisite demands one to understand the rates, volumes and propensity for those conversion and reactions, and what the impact is on other dietary components. These details are critical and are often brushed aside. 

Certainly, there is a massive amount of evidence that nitrosamines can cause cancer. That is not in question. However, that statement does not hold true for nitrate or nitrite. This will be clearer when we explore the biochemistry of how we form a nitrosamine.

Getting back to basics, we need to remind ourselves that chemical reactions need to be balanced. Each side of the equation must be equal in charge and in the number of atoms. We cannot arbitrarily, lose or gain, charge or mass. Further, the direction of the reaction is driven by the energy required. Apologies for the high school primer but it is worthwhile mentioning here.

An amine e.g., R-NH2 can be the substrate to form a nitrosamine, R-NHNO. What is required is that a proton (H+) is replaced by a nitroso group (NO+). This keeps the reaction balanced in terms of charge. Here is an example of the reactions to form a nitrosamine.

R-NH2 + NO+ -----> R-NHNO + H+ 

The equation is balanced, there are an equal number of atoms and charge on both sides. What is formed is a nitrosamine and a proton, which will then react to an OH- to form water.

Now if you replace the nitrosonium (NO+) group with nitrite (NO2-) you will see that the reaction will not balance. The problem is that nitrite has a negative charge vs. a positive charge for nitrosonium, plus there is an extra oxygen. Simply put, nitrite is not a nitrosating species, but other reactive nitrogen species are. This is matched by animal toxicity studies that fail to find any effects of nitrate on carcinogenesis or mutagenesis (17, 18).

There is some similar chemistry where the end result is a nitration reaction as opposed to a nitrosation. These are also common in states of inflammation & immune activation (17) and result in the replacement of a proton in the amine with NO2+ vs the NO+ in nitrosation. If for example the substrate is the amino acid tyrosine, the end result would be nitrotyrosine. Formation of nitrotyrosine tracks with states of infection, immune activation and inflammation, and are linked to a number of dysfunctional outcomes including cancer (3, 14, 17-20).

What Reactive Nitrogen Species can form a Nitrosamine?

Specifically, you need a donor of a nitroso group (NO+) and certainly nitrite and nitrate do not qualify. Interestingly, you can start with nitric oxide, but you need lots of it, and you end up with smog, the brown gases nitrogen dioxide NO2, or dinitrogen trioxide N2O3.  

2NO + O2  ----> 2NO2

Without getting too bogged down in complex chemistry, this reaction follows 2nd order kinetics, so with respect to the concentration of NO the generation of smog is based on the square of the NO concentration. In essence the more NO you have the rate of production of the smog gas NO2 is accelerated at the square of NO concentration. If levels of NO are raised 10-fold then the reaction to form NO2 is accelerated 100-fold. If the NO levels are raised 100-fold then the NO2 production is now 10,000 times greater. This exponential increase in NO2 production outlines how generation of NO by iNOS is the driver of the problem and not the relatively minor conversion of nitrite to nitric oxide.

A critical additional consideration is that both NO and oxygen being more soluble in lipids than water, so their relative concentration is higher in lipids. My lab documented that the reaction is accelerated over 300x in lipids like membranes. Indeed, it is estimated that 90% of this reaction to form smog occurs in membranes of cells (21). 

You will notice that the smog gas, nitrogen dioxide (NO2), also looks a lot like nitrite (NO2-), the difference is that nitrite has an additional electron, and what a difference this makes. Nitrite is water soluble and quite stable, whereas nitrogen dioxide is lipid soluble and very reactive. The smog gas, nitrogen dioxide, also forms its related smog gas N2O3, quite easily via the reaction of an additional nitric oxide with NO2. 

NO + NO2 -----> N2O3 

Either nitrogen dioxide or dinitrogen trioxide can then readily form a nitrosamine, the cancer evoking moiety that is at the center of the controversy. 

NO2 + R-NH2 ----> R-NHNO + H2O

N2O3 + R-NH2  R-NHNO + NO2 

If you are connecting the dots that the high levels of smog also increase the risk for lung cancer, then you are on the right path. A critical chemical link between smog and cancer are these two brown gases. The same chemistry comes into play when the lungs become inflamed by inhaling particular matter, as then the iNOS isoform of nitric oxide synthase is expressed and large amounts of NO are produced from endogenous sources. In turn, these are converted to reactive nitrogen species like smog gases.

In short, dietary nitrate and nitrite do not provoke cancer via nitrosamines because they are:

• too stable
• the wrong charge 

? must be converted to other reactive nitrogen species

• the rates that nitrate and nitrite are converted to nitric oxide can support cardiovascular functional optimization but are inadequate for nitrosamine formation, for the same reasons that endogenous production from either nNOS or eNOS do not support nitrosamine formation, and neither does saliva.

The Population Studies on Diet & Rulings from the IARC/WHO

Few appreciate that World Health Organization (WHO) guidelines are generated from rubber stamping recommendations from the IARC. The IARC is most concerned about the potential link between dietary nitrate/nitrite and stomach cancer based on the conversion of nitrite to nitrous acid (via gastric acid) and then to nitrosating species (22-23). This is based on animal studies and the use of simple acid-amine-nitrite systems.

Food, particularly food-borne antioxidants, can readily suppress the formation of nitrosamines. Few appreciate that there is an active pump that transports plasma vitamin C into the gastric lumen to interact with stomach acid (24). Thus, antioxidants may interfere with these reactions from sources within ingested food or from endogenous sources pumped into the stomach lumen. 

Mixed messages have arisen from the IARC, in their 2006 report they noted that food and water sources of nitrate & nitrite as drivers for cancer were not supported by epidemiology studies (25). Indeed, the IARC noted numerous studies that evaluated dietary intake of nitrate and gastrointestinal cancer, specifically gastric, and noted that in each one of these large cohort studies the relationship was an inversive. In other words, diets high in nitrate were associated with LOWER rates of cancer, NOT higher (4-8). These studies, which exhibit the polar opposite to the IARC edict, are attributed to the benefits of fruits and vegetables. Now we also appreciate that the gut microbiome is also an important contributor.

Some years later, the IARC was still sticking to its general conclusions by stating that “Ingested nitrate or nitrite under conditions that result in endogenous nitrosation is probably carcinogenic to humans.” 

In other words, the IARC maintains the tone of its conclusion, but only to reiterate its validity in a theoretical state, while acknowledging that the epidemiology does to support these conclusions. The end result of this scientific bias is confusion and misconception as lay press headlines persist in reiterating the inaccuracies. These inaccuracies gain more reads, clicks and sells. By contrast, scientists and other esteemed societies have noted that the concerns related to nitrate and nitrite and cancer have been overstated (26). 

Conclusion

While the IARC has stuck to its platform in the theoretical, they acknowledge that there is no clear epidemiology to support this association. Rather the opposite tends to be true, plant-based diets that are rich in nitrate are associated with lower cancer risk, as well as enhanced cardiovascular health. Given that the chemistry is well documented, that nitrate or nitrite are not nitrosating species, and that the formation of nitrosamines is dictated by other reactive nitrogen species, it is clear that these concerns have been overstated for some decades. What is likely to be a better explanation is that endogenous production of reactive nitrogen species like NO2, N2O3 and peroxynitrite resulting from chronic inflammation and activation of the immune response, is responsible.

We should move on from the hypothetical risks of ingested nitrate/nitrite and cancer. Indeed we are doing a disservice to the community by not promoting diets rich in nitrate/nitrite for cardiovascular health and wellness in general.

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