A Review of the in Vivo Evidence Investigating the Role of Nitrite Exposure from Processed Meat Consumption in the Development of Colorectal Cancer

nutrients

Discussion A Review of the In Vivo Evidence Investigating the Role of Nitrite Exposure from Processed Meat Consumption in the Development of Colorectal Cancer

William Crowe, Christopher T. Elliott and Brian D. Green * Institute of Global Food Security, School of Biological Sciences, Queens University Belfast, Belfast BT9 5DL, UK; [email protected] (W.C.); [email protected] (C.T.E.) * Correspondence: [email protected]; Tel.: +44-(0)28-90976541

Received: 14 August 2019; Accepted: 14 October 2019; Published: 5 November 2019

Abstract: The World Cancer Research Fund (WCRF) 2007 stated that the consumption of processed meat is a convincing cause of colorectal cancer (CRC), and therefore, the public should avoid it entirely. Sodium nitrite has emerged as a putative candidate responsible for the CRC-inducing eﬀects of processed meats. Sodium nitrite is purported to prevent the growth of Clostridium botulinum and other food-spoiling bacteria, but recent, contradictory peer-reviewed evidence has emerged, leading to media reports questioning the necessity of nitrite addition. To date, eleven preclinical studies have investigated the eﬀect of consuming nitrite/nitrite-containing meat on the development of CRC, but the results do not provide an overall consensus. A sizable number of human clinical studies have investigated the relationship between processed meat consumption and CRC risk with widely varying results. The unique approach of the present literature review was to include analysis that limited the human studies to those involving only nitrite-containing meat. The majority of these studies reported that nitrite-containing processed meat was associated with increased CRC risk. Nitrite consumption can lead to the formation of N-nitroso compounds (NOC), some of which are carcinogenic. Therefore, this focused perspective based on the current body of evidence links the consumption of meat containing nitrites and CRC risk.

Keywords: nitrite; processed meat; colorectal cancer

1. Introduction It has been reported that between 66% and 99% of Europeans consume processed meat, with the mean amount consumed per day ranging between 10 and 80 g per day [1]. Processed meats are defined as meats that have been modified through curing, fermentation, salting, smoking, or otherwise, for the purpose of improving shelf life and/or enhancing flavour. Red meat is a nutritionally important source of protein, providing all essential amino acids and minerals, such as iron, selenium, and zinc. In 2007, the World Cancer Research Fund (WCRF) stated that there is convincing evidence linking the consumption of red and processed meat with the development of colorectal cancer (CRC) [2]. The WCRF further stated that the public should limit their intake of red meat to below 500 g per week, and avoid processed meat entirely. A further update from the WCRF emphasized that no safe level of processed meat could confidently be attributed to a lack of risk [3]. The International Agency for Research on Cancer (IARC) stated that with every increase of 50 g of processed meat consumption per day, the risk of CRC rises by 18%, whilst with every increase of 100 g of red meat consumed per day the risk of CRC rises by 17% [4]. Numerous meta-analyses have been conducted in this area, the majority of which have reported processed meat to be linked to CRC development [5–12]. Due to the narrow

Nutrients 2019, 11, 2673; doi:10.3390/nu11112673 www.mdpi.com/journal/nutrients Nutrients 2019, 11, x FOR PEER REVIEW 2 of 17 Nutrients 2019, 11, 2673 2 of 17 development [5–12]. Due to the narrow scope of a meta-analysis, no animal evidence is included in the synthesis,scope of a but meta-analysis, animal studies no animalare well evidence controlled, is included so it is important in the synthesis, to consider but animalthem in studies the balance are well of evidence.controlled, so it is important to consider them in the balance of evidence. Genetic riskrisk factors factors are are undoubtedly undoubtedly involved involved in CRC in development;CRC development; however, however, increasing increasing evidence evidencesuggests asuggests more minor a more role minor than previously role than thought.previously Studies thought. mapping Studies the mapping geographical the geographical incidences of incidencescancer have of noted cancer that have incidences noted that in incidences immigrants in beginimmigrants to reflect begin that to ofreflect the host that nations’of the host incidences nations’ withinincidences a single within generation a single [13generation], indicating [13], that indi environmentalcating that environmental factors are the factors largest are contributor the largest to contributorCRC development. to CRC development. A number of of components components present present in in processed processed meat meat have have been been implicated implicated as aspotential potential causes causes of CRCof CRC including including heterocyclic heterocyclic amines amines (HCA), (HCA), polycyclic polycyclic hydrocarbons hydrocarbons (PAH), (PAH), nitrites, nitrites, haem haem iron, iron,and highand highfat. HCA fat. HCAand PAH and PAHare also are present also present in other in otherfoodstuffs foodstu thatffs are that not are associated not associated with CRC, with CRC,such assuch fish as and fish poultry, and poultry, and therefore, and therefore, have been have largel beeny largely ruled out ruled [14]. out Nitrites [14]. Nitrites have emerged have emerged as a leading as a candidateleading candidate responsible responsible for processed for processed meats’ meats’ associat associationion with withCRC; CRC; however, however, it is it possible is possible that that a combinationa combination of ofthe the constituents constituents listed listed could could coalesce coalesce to initiate to initiate the thepathogenesis pathogenesis of CRC. of CRC. Nitrites Nitrites are anare effective an effective preservative preservative that that uniquely uniquely prevent prevent the the growth growth of of clostridiumclostridium botulinum botulinum.. Nitrites Nitrites also enhance the colour and flavourflavour of processed meat. Nitrite consumpt consumptionion can lead to the endogenous formation of N-nitroso compounds (NOC), some ofof whichwhich areare carcinogeniccarcinogenic [[15].15]. The WCRFWCRF hashas extensively extensively reviewed reviewed the the existing existing research research concerning concerning the etheffect effect of processed of processed meat meatconsumption consumption and CRC and andCRC their and conclusions their conclusions appear appear to be definitive. to be definitive. There isThere good is reason good toreason believe to believethat a causal that a relationship causal relationship exists; however, exists; however, there is not there consensus is not consensus of opinion of in opinion the scientific in the literature.scientific Thisliterature. review This analyses review the analyses conflicting the preclinicalconflicting andprec clinicallinical and research clinical investigating research investigating the role of processed the role ofmeat processed consumption meat inconsumption the development in the of CRC.development There is aof specific CRC. There focus onis thea specific relationship focus between on the relationshipnitrite-containing between processed nitrite-containing meat and CRC. processed meat and CRC.

1.1. Methods Methods As shownshown inin Figure Figure1, a1, literature a literature search search was conductedwas conducted using 3using search 3 engines:search engines: Pubmed, Pubmed, Scopus, Scopus,and Web and of Science.Web of Science. A combination A combination of the followingof the following terms wereterms searched: were searched: colorectal colorectal cancer, cancer, CRC, processedCRC, processed meat, meat, sausage, sausage, bacon, bacon, nitrite, nitrite, and N and-nitroso N-nitroso compounds. compounds.

Figure 1. SearchSearch strategy and results, including reasons for exclusion. 1.2. Inclusion and Exclusion 1.2. Inclusion and Exclusion Studies were included provided that they met the following criteria: full text was available, writtenStudies in English, were included including provided single or that multiple they measuresmet the following of colorectal criteria: cancer; full that text it was was available, a human writtenstudy that in English, included including a measurement single ofor processedmultiple measures meat consumption. of colorectal Reviews cancer; andthat technical it was a reportshuman werestudy excluded. that included Studies a measurement that only utilised of processed in vitro techniquesmeat consumption. were excluded. Reviews and technical reports were excluded. Studies that only utilised in vitro techniques were excluded. Nutrients 2019, 11, 2673 3 of 17

1.3. Results The search yielded 2478 results, of which 61 were deemed to be appropriate. A total of 2257 articles were excluded owing to duplication; ﬁve articles were not in English; full text was not available for 31 articles; 23 reviews/technical reports/letters to editors were excluded; 16 studies used only in vitro techniques; 85 human studies did not measure processed meat consumption.

2. Preclinical Evidence As shown in Table1, eleven preclinical studies have investigated the e ffect of nitrite-containing processed meat consumption on CRC development. Eight studies employed Fischer rats, one employed Sprague Dawley rats, one employed ApcMin mice, and one used A/J mice and CF-1 mice. Unfortunately, the ApcMin study exposed animals to nitrite supplemented water, and therefore, did not supplement processed meat with nitrite. In nine of the studies, the development of CRC was confirmed by the occurrence of aberrant crypt foci (ACF) or mucin depleted foci (MDF). One study measured faecal levels of NOC and one measured total N-nitroso compounds, cytotoxicity, and thiobarbituric-acid-reactive substances. Three of the eleven studies concluded that nitrite exposure did not increase the risk of CRC [16–18]. One found that hot dog did not increase the risk but 1.5 g sodium nitrite/L did [19]. Five studies concluded that nitrite exposure resulted in an increased risk of CRC [20–24]. Mirvish et al. 2003 reported that nitrite consumption increased NOC excretion but did not measure any CRC outcomes [25]. One study reported that nitrite ingestion elicited a protective effect on CRC development [26]. Those studies which found that nitrite-containing processed meat was causative for CRC, all exposed animals to a dietary amount of at least 50% processed meat. Although potentially achievable in the human diet, it seems unrealistic. Conversely, the only study which found processed meat to be protective for CRC provided a 60% meat diet for a period of 100 days. One explanation for the different outcome of this study is that the processed meat (bacon) group contained much higher NaCl levels than the control. This increases water intake and subsequently raises faecal moisture content. The intestinal dilution of nitrous compounds is one possibility for the reported findings. Two studies that reported a causative relationship did not have adequate control groups to conclude that the findings were due to nitrite [22,23]. Furthermore, neither had a negative control, and were both are unable to conclude that processed meat increases the risk of CRC compared with a meat-free diet. Two studies reporting no effect used nitrite-containing water rather than nitrite in a food matrix [16,23]. In these studies, nitrite and haem iron were provided concomitantly, but not protein. The carcinogenic NOCs form most efficiently in the presence of high levels of nitrite, iron, and protein [27]. Therefore, studies which add nitrite alone or in the absence of either iron and protein, will not realise the full carcinogenic effect. The above two studies were also the only studies not to actually measure ACF and MDF. The majority of studies investigating nitrite causation of CRC measured ACF and MDF as the primary outcomes. Adenomas typically take several months to manifest, whilst ACF and MDF develop in a matter of weeks. Therefore, ACF and MDF are used widely used, especially as there is a high concordance between the quantity of ACF and MDF and the development of adenomas. ACF is an early histopathological indicator of CRC, but it should be pointed out that not all ACF lesions lead to clinical CRC. These are easily identifiable pre-neoplastic lesions that are typically present on the mucosal surface. MDF are a subset of ACF, the primary difference being that mucous production is suppressed in MDF. Visual inspection of the colon remains the gold standard for confirming the determination of disease pathology. Although there are putative blood, urinary, and faecal biomarkers, none of them are as specific and sensitive as ACF and MDF [28,29]. Nutrients 2019, 11, 2673 4 of 17

Table 1. Characteristics of animal studies assessing colorectal cancer and nitrite consumption

Author Model Intervention Fat Content Control Outcome 30% bacon (freeze dried), 70% AIN-76 for AIN-76 formula with identical Fecal NOC level ↑ [17] Parnaud et al. 1998 Fischer rat 100 days 7%, 14%, 28% protein and fat (Casein and lard No ACF were detected in the colon 60% bacon for 30 days used to increase macros) of bacon-fed uninitiated rats. 30% bacon (freeze dried), 70% AIN-76 for AIN 76 formula with identical ACF by 12% in rats fed a diet with 14% ↓ [26] Parnaud et al. 2000 Fischer rat 100 days protein and fat (casein, olive oil 30% bacon and by 20% in rats fed a 28% 60% bacon for 100 days and lard used to increase macros) diet with 60% bacon. TD-01407 Sprague– Dawley rats TD-98061 18% hot dog, 82% TD-01407 SP 7 days Fecal NOC in hot dog and beef [25] Mirvish et al. 2003 and male mice of 26 g/100 g AIN-76A diet ↑ 15% beef. 82% TD-01407 SP 7 days fed compared with control. various strains soy oil and casein added to increase macros [20] Santarelli et al. 2010 Fischer rat 55% processed pork (moist), 45% AIN76 15 g/100 g AIN-76A MDF in processed pork group. ↑ ACF and MDF in cured ham [21] Pierre et al. 2010 Fischer 344 rats 55% cured ham (freeze dried), 45% AIN76A - Ain-76A ↑ fed group. MDF in hot dog fed group. 55% processed meat (moist), 40% AIN76, 5% ↑ [21] Santarelli et al. 2013 Fischer rat 30% AIN-76A with 5% saﬄower Addition of calcium carbonate saﬄower oil suppresses lesions. AIN-76 with sodium nitrite in drinking [16] Chenni et al. 2013 F344 rats water (1 g/L) - AIN-76A No change. nitrite (0.17 g/L) and nitrate (0.23 g/L) MDF in cured meat fed group, 55 g (moist weight) experimental cured meat, ↑ [22] Pierre et al. 2013 Fischer 344 rats 15% AIN-76A compared with vitamin E and 45 g AIN76 100 days calcium supplemented groups. F344 ratsC57BL/6J (0.17 g/L of NaNO and 0.23 g/L of NaNO ) 2 3 MDF in heme iron fed group. [18] Bastide et al. 2015 ApcMin/+ added to water - AIN-76A ↑ No change in nitrite fed group. miceApc+/+ mice AIN-76A with 2.5% haemoglobin

0.5 or 1.0 g NaNO2/L No change following hot dog [19] Zhou et al. 2015 A/J mice CF-1 mice 0, 1.0, 1.25, or 1.5 g NaNO /L - AIN93G ingestion. ACF in 1.5 g compared 2 ↑ 18% hot dog with untreated 50 g cooked, cured meat, 50 g AIN76 [23] Bastide et al. 2017 Fischer 344 rats 100 days - Ain-76A MDF in cured meat fed group. ↑ vs polyphenol rich diet increased; decreased; ACF Aberrant crypt foci; CRC colorectal cancer; NOC N-nitroso compound level; MDF mucin depleted foci. ↑ ↓ Nutrients 2019, 11, 2673 5 of 17

In summary, the existing preclinical research investigating the effect of nitrite exposure on CRC development is conflicting and methodological inconsistencies are apparent. Furthermore, the lack of a dose-response study is a major deficiency. The heterogeneous nature of chemical-induced cancer in animal models can be problematic, due to the unpredictability of the location and timing of cancer development [30]. Despite the variability that ensues, the majority of existing studies have used chemically induced models. Future studies should investigate models spontaneously developing CRC, such as the ApcMin mouse. This is a murine model that is susceptible to colorectal adenomas, in which there is a deletion of the APC gene. It is similar to the human hereditary condition, familial adenomatous polyposis (FAP). The use of in vivo models with similar pathogeneses to human conditions will increase the ability to extrapolate the findings. Differences in physiological processes exist between humans and murine models, preventing direct extrapolation of findings. For example, the microflora of humans and rats differ and given that bacteria can increase the formation of nitrosamines [31]. Such differences may have a considerable effect on the association between nitrite consumption and CRC development. It is, therefore, imperative to give greater credence to human studies.

3. Clinical Evidence A large number of studies of differing designs have investigated the relationship between human processed meat consumption and CRC. Table2; Table3, respectively, describe all prospective studies and case-control studies conducted thus far. Supplementary Tables S1 and S2 include more information about the studies presented in Tables2 and3. Of the 49 human studies identified, 23 found that processed meat consumption was linked with CRC [32–54], 25 found no link [55–79], and one study found processed meat to be protective for CRC [80]. It is, therefore, difficult to draw any definitive conclusions from these studies. Nutrients 2019, 11, x FOR PEER REVIEW 7 of 17

Table 3. Characteristics of case control human studies assessing colorectal cancer and processed meat consumption

Author Cases (n =) Controls (n =) Description of processed meat Relative Risk (CI) Findings [53] Lohsoonthorn et al. 1995 279 279 Bacon 12.49 (1.68–269.1) Significantly ↑ bacon consumption in cases group [54] De Stefani et al. 2012 321 844 Processed meat 3.53 (1.93–6.46) 2.01 (1.07–3.76) Significantly ↑ risk of CRC Nutrients 2019, 11, 2673[34] Tajima and Tomina 1985 93 186 Ham and sausage 2.87 Significantly ↑ risk of CRC6 of 17 [35] Levi et al. 2004 323 1271 Processed meat 2.53 (1.50–4.27) Significantly ↑ risk of CRC [36] Haenszel et al. 1973 179 357 Sausage and other processed pork 2.30Ϟ 1.77Ф 2.7҂ Significantly ↑ risk of CRC [37] YoungTable and Wolf 2. 1988Characteristics 152 ⱡ of201 prospective ‡ 618 human studies Processed assessing lunch colorectal meat cancer and 1.85processed (1.33–2.58) meat consumption Significantly ↑ risk of CRC [38] Bidoli et al. 1992 123¥ 125₸ 699 Salami and sausages 1.8¥ 1.9₸ Sig ↑ ¥ but not ₸ [39] Navarro et al. Sample2003 Colorectal 287 Description 566 of Cold cuts and sausages 1.64 (1.16–2.32) Significantly ↑ risk of CRC Author Relative Risk (CI) Findings [40] Rosato et al. 2013Size Cancer 329 Cases Processed 1361 Meat Processed meat 1.56 (1.11–2.20) Significantly ↑ risk of CRC [41] Hu et al. 2008 3174 5039 Processed meats 1.50 (1.2–1.8) Significantly ↑ risk of CRC [32] Oba et al. 2005 31,552 213 Processed meat 1.98 (1.24–3.16) 0.85 (0.50–1.43) Significantly risk of CRC in not [60] Williams et al. 2010 945 959 Processed meat 1.36 (0.80–1.68)Ւ 1.02 (0.38–1.96)ⱦ ↑ No significant risk of CRC [33] Goldbohm et al. 1994 3123 393 Processed meat 1.72 (1.03–2.87)♂ ♀ Significantly risk of CRC♂ ♀ [61] Benito et al. 1990 286 498 Processed meat 1.36 ↑ No significant risk of CRC [44] Parr et al. 2013 84,210 674 Processed meat 1.54 (1.08–2.19) Significantly risk of CRC ↑ [42] De Verdier et al. 1991 559 505 Sausage 1.30 (0.8–1.9)¥ 1.7 (1.1–2.8)₸ ↑ Significantly risk of CRC [48] Wu et al. 2006 14,032 581 Processed meat 1.52 (1.12–2.08) Significantly risk of CRC [62] Dales et al. 1978 99‡ 280 Nitrite treated meats 1.22 ↑ No significant risk of CRC [55] Bostick et al.[63] 1994 Joshi et al. 2015 35,215 212 3350 Processed 3504 meat Processed 1.51 meats (0.72–3.17) 1.20 (1.0–1.4) No significant risk of CRC No significant risk of CRC [49] English et al.[64] 2004 Balder et al. 2006 37,112 451 1535 Processed 4371 meat Processed 1.50 meats (1.1–2.0) 1.18 (0.84–1.64) Significantly risk of CRC No significant risk of CRC ↑ [50] Norat et al.[65] 2005 Murtaugh et al. 478,040 2003 1329 952 Processed 1205 meat Processed 1.42 meat (1.09–1.86) 1.18 (0.87–1.61) ♀ 1.23 (0.84–1.81) Significantly ♂ risk of CRCNo significant risk of CRC ↑ [56] Takachi et al.[66] 2011 Kimura et al. 2007 98,514 1145 782 Processed 793 meat 1.27 Processed (0.95–1.71) meats1.19 (0.82–1.74) 1.15 (0.83–1.60) No significant risk of CRC No significant risk of CRC [51] Willet et al.[68] 1990 Nothlings et al. 88,751 2009 150 1009 Processed 1522 meat Processed 1.21 meat (0.53–2.72)♂ ♀ Significantly 1.08 (0.89–1.39) risk of CRC in the 3rd quintile No significant but not therisk 4th of CRC ↑ [67] Pietinen et al.[69] 1999 Steinmetz and 27,Potter 111 1993 185 220 Processed 438 meat Processed 1.20 meat (0.7–1.8) 1.03 (0.55–1.95)♂ 0.77 (0.35–1.68) No significant♀ risk of CRCNo significant risk of CRC [52] Cross et al.[70] 2007 Franceschi et al. 494,036 1997 5107 1225 Processed 4154 meat Processed 1.20 meat (1.09–1.32) 1.02 (0.89–1.24) Significantly risk of CRC No significant risk of CRC ↑ [46] Bradbury et[71] al. Centozone 2019 2009 468,910 2576 119 Processed 119 meat Processed 1.19 meat (1.01–1.41) 1.01 Significantly risk of CRC No significant risk of CRC ↑ [73] Giovannucci[72] et Tiemersma al. 1994 et al. 47,949 2002 205 102 Processed 537 meat Sausage 1.16 (0.44–3.04) 0.90 (0.6–1.3) No significant risk of CRC No significant risk of CRC [47] Cross et al.[43] 2010 Macquart-Moulin 300,948 1986 2719 399 Processed 399 meat Charcuterie 1.16 (1.01–1.32) 0.89 Significantly risk of CRC Significantly ↑ risk of CRC ↑ [74] Chao et al.[80] 2005 Iscovich et al. 1992 148,610 1197110 Processed 220 meat Processed 1.13 meat (0.91–1.41) 0.43 (0.21–0.89) No significant risk of CRC Significantly ↓ risk of CRC [75] Lee et al. 2009[45] Nowell et al. 2002 74,942 394 157 Salted 380 meat Sausage and 1.10 bacon (0.8–1.4) - No significant Significantly risk of CRC ↑ bacon consumption in cases group [76] Larsson et al. 2005↑ increased; 61,433 ↓ decreased234 ⱡ 155 Proximal Processedcolon; ‡ distal meat colon; ♂ male; 1.07♀ female; (0.85–1.33) ¥ colon; ₸ rectal; Ւ caucasion; No ⱦ significant African American; risk of CRC CRC colorectal cancer. Where ‡ [77] Ollberding et al.the 2012 authors 215,000did not provide the 3404 relative risks Processed for males meat and females combined, 1.06 (0.94–1.19) we have provided the gender specific No relative significant risks. risk Relative of CRC risk values reflect those in the [78] Egeberg et al. 2013highest consumption 53,988 group vs 914 those in the Processed lowest consumption meat group. 1.02 All (0.78–1.34) relative risk values are adjusted, with Nothe significantexception o riskf Iscovich of CRC et al and Nowell et al. more [79] Flood et al. 2002 45,496 487 Processed meat 0.97 (0.73–1.28) No significant risk of CRC details of adjustments are provided in supplementary table 2. [57] Sato et al. 2006 47,605 358 Ham or sausage 0.91 (0.61–1.35) No significant risk of CRC [58] Lin et al. 2004 37,547 202 Processed meat 0.85 (0.53–1.35) No significant risk of CRC [59] Knekt et al. 1999 9985 73 Nitrite 0.74 (0.34–1.63) No significant risk of CRC male; female increased; Proximal colon; distal colon; CRC colorectal cancer. Where the authors did not provide the relative risks for males and females combined, we have ↑ ‡ provided♂ ♀ the gender specific relative risks. Relative risk values reflect those in the highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, more details are provided in Supplementary Table S1. Nutrients 2019, 11, x FOR PEER REVIEW 7 of 17 Nutrients 2019, 11, x FOR PEER REVIEW 7 of 17 Table 3. Characteristics of case control human studies assessing colorectal cancer and processed meat consumption Table 3. Characteristics of case control human studies assessing colorectal cancer and processed meat consumption Author Cases (n =) Controls (n =) Description of processed meat Relative Risk (CI) Findings Author Cases (n =) Controls (n =)[53] Lohsoonthorn DescriptionNutrientsNutrients 2019 et of2019 al. processed, 111995, 11, x, FORx FOR meat PEER PEER 279 REVIEW REVIEW Relative 279 Risk (CI) Bacon Findings 12.49 (1.68–269.1)7 7of of17 17 Significantly ↑ bacon consumption in cases group [53] Lohsoonthorn et al. 1995Nutrients 2019, 27911Nutrients, x FOR PEER 2019 279,REVIEW 11 , x FOR[54] PEERDe Stefani REVIEW et al. Bacon 2012 321 12.49 844 (1.68–269.1) 10 Processed of 17 Significantly meat 10 ↑of 17 bacon consumption 3.53 (1.93–6.46) in cases 2.01 (1.07–3.76)group Significantly ↑ risk of CRC [54] De Stefani et al. 2012 321 844 [34] Tajima and Processed Tomina 1985 meat 93 3.53 (1.93–6.46) 186 2.01 (1.07–3.76) Ham and sausage Significantly ↑ risk of CRC 2.87 Significantly ↑ risk of CRC TableTable 3. 3.Characteristics Characteristics of of case case control control human human studies studies assessing assessing colorectal colorectal cancer cancer and and processed processed meat meat consumption consumption [34] Tajima and Tomina 1985 93 186 [35] Levi et al. Ham 2004 and sausage 323 1271 2.87 Processed meat Significantly ↑ risk of 2.53 CRC (1.50–4.27) Significantly ↑ risk of CRC [35] Levi et al. 2004 323 1271Table 4.[36] Characteristics HaenszelTable et Processed al.4. of1973Characteristics Authorprospective Author meat and of 179prospective case Cases controlCases (n and (=)n 2.53human 357 =) case (1.50–4.27) ControlsControls studiescontrol Sausage( n ( =)nassessinghuman =) and Description studies Descriptionother colorectal processed assessing of of cancerprocessed Significantly processed pork colorectal and meat nitritemeat ↑ cancer risk containing 2.30of andCRCϞ 1.77 nitrite Relative Ф Relativemeat 2.7 ҂containing Risk Risk (CI) (CI) meat Significantly ↑ risk Findings Findingsof CRC ↑ [36] Haenszel et al. 1973 179 357 [37] Young Sausage[53] [53]and Lohsoonthornand LohsoonthornWolf other 1988 processed et etal. al. 1995 pork 1995 152 ⱡ 201 ‡ 279 279 2.30 618Ϟ 1.77Ф 279 2.7 279 ҂ Processed lunch meat Bacon BaconSignificantly ↑ risk of 1.85 CRC (1.33–2.58) 12.49 12.49 (1.68–269.1) (1.68–269.1) Significantly Significantly ↑ ↑ bacon baconrisk ofconsumption consumptionCRC in incases cases group group Author Sample size Colorectal cancer cases Description of processed meat Relative Risk (CI) Findings ↑ [37] Young and Wolf 1988 152Author ⱡ 201 ‡ 618 Sample [38] size Bidoli Colorectal[54] et[54] Processed al. De De1992 Stefani cancer Stefani lunch etcases etal. meatal. 2012 2012 123¥ Description 125₸ 321 321 of 1.85processed 699 (1.33–2.58) meat 844 844 Salami andRelative sausages Processed Processed Risk Significantly meat (CI)meat ↑ risk of 3.53 CRC 1.8¥ 3.53 (1.93–6.46) 1.9(1.93–6.46)₸ Findings 2.01 2.01 (1.07–3.76) (1.07–3.76) Sig Significantly Significantly¥ but not ₸↑ ↑ risk risk of ofCRC CRC [48] Wu et al. 2006 14,032 581 ‡ Sausage, salami, bologna 1.52 (1.12–2.08) ↑ Significantly ↑ risk of CRC ↑ [38] Bidoli et al. 1992 [48] Wu et al. 123¥ 2006 125 ₸ 699 14,032[39] Navarro[34][34] Salami etTajima al.Tajima 581 2003 and‡ and and sausages Tomina Tomina 1985 1985 287 Sausage, 93 93salami, 566 1.8¥ bologna 1.9₸ 186 186 Cold cuts and Ham 1.52 Hamsausages and (1.12–2.08) and sausage sausageSig ↑ ¥ but not 1.64 ₸ (1.16–2.32) Significantly 2.87 2.87 risk of CRC Significantly Significantly Significantly risk of ↑ CRC ↑ risk risk of ofCRC CRC Salami, continentalSalami, sausages, continental sausages sausages, or sausages or ↑ ↑ [39] Navarro et al. 2003 [49] English et 287al. 2004 [49] English et 566 al.37,112 2004[40] Rosato[35] Cold [35]et37,112 Levial. Levi cuts2013 284 et etandal. al. 2004 sausages2004 284 329 323 323 1.64 1361 (1.16–2.32) 1271 1271 Processed Processed meat1.50 Processed (1.1–2.0) Significantly meat meat 1.50 (1.1–2.0) risk of 1.56 CRC (1.11–2.20) Significantly 2.53 2.53 (1.50–4.27) (1.50–4.27) ↑ risk Significantly of CRC ↑ risk Significantly of CRC Significantly Significantly risk of ↑ CRC ↑ risk risk of ofCRC CRC frankfurters, bacon,frankfurters, ham bacon, ham ↑ ↑ [40] Rosato et al. 2013 329 1361 [41] Hu et[36] al.[36] 2008 Haenszel Processed Haenszel et meatetal. al. 1973 1973 3174 179 179 1.56 5039 (1.11–2.20) 357 357 Processed Sausage Sausage and meats and other other Significantly processed processed pork pork risk of 1.50 CRC (1.2–1.8) 2.30 2.30Ϟ Ϟ1.77 1.77Ф Ф2.7 2.7҂ ҂ SignificantlySignificantlySignificantly risk of ↑ CRC ↑ risk risk of ofCRC CRC [56] Takachi et al. 2011[56] Takachi et al. 98,514 2011 98,514 1145 1145 Processed meat Processed meat 1.27 (0.95,1.71) ♂ 1.191.27 (0.82,1.74) (0.95,1.71) ♀↑ ♂ 1.19 (0.82,1.74)No ♀ significant risk Noof CRC significant risk of CRC [41] Hu et al. 2008 3174 5039 [60] Williams[37][37] Younget Processed Youngal. 2010 and and meats Wolf Wolf 1988 1988 945 152 152 ⱡ 201 ⱡ 201 ‡1.50 959 ‡ (1.2–1.8) 618 618 Processed Processed Processed meat lunch Significantlylunch meat meat 1.36 risk(0.80–1.68) of CRCՒ 1.02 1.85 1.85 (0.38–1.96) (1.33–2.58) (1.33–2.58)ⱦ No significant Significantly Significantly risk of CRC↑ ↑ risk risk of ofCRC CRC [57] Sato et al. 2006[57] Sato et al. 2006 47,605 47,605 358 358 Ham or sausage Ham or sausage 0.91 (0.61–1.35) 0.91 (0.61–1.35) No significant risk Noof CRC significant risk of CRC [60] Williams et al. 2010 945 959 [61] Benito[38] et[38] al.Bidoli Processed Bidoli1990 et etal. al. 1992meat 1992 286 123¥1.36 123¥ (0.80–1.68)125 125₸ 498₸ Ւ 1.02 699 (0.38–1.96) 699 ⱦ Processed Salami Salami meat and and sausages No sausages significant risk of CRC 1.36 1.8¥ 1.8¥ 1.9 1.9₸ ₸ No significantSig riskSig ↑ ↑ of¥ CRC¥ but but not not ₸ ₸ [59] Knekt et al. 1999[59] Knekt et al. 1999 9,985 9,985 73 73 Nitrite Nitrite 0.74 (0.34–1.63) 0.74 (0.34–1.63) No significant risk Noof CRC significant risk of CRC [61] Benito et al. 1990 286 498 [42] De Verdier[39] Processed Navarroet al. 1991 etmeat al. 2003 559 287 505 1.36 566 Sausage Cold cuts and No sausages significant 1.30risk (0.8–1.9)¥of CRC 1.7 1.64(1.1–2.8) (1.16–2.32)₸ Significantly Significantly ↑ risk of↑ CRC ↑ risk of CRC Author Author Cases (n =) [39]CasesControls Navarro (n =) ( n et=) al. 2003Controls (n Description =) 287 of processed Description meat 566 of processed meat Cold Relative cuts and Risk sausages (CI) Relative Risk (CI) 1.64 (1.16–2.32) Findings Findings Significantly risk of CRC [42] De Verdier et al. 1991 559 505 [62] Dales et[40] al. 1978Rosato Sausage et al. 2013 99 1.30 329 (0.8–1.9)¥ 280 1.7 (1.1–2.8) 1361 ₸ Nitrite treated Processed meats Significantly meat ↑ risk of CRC 1.22 1.56 (1.11–2.20) No significant Significantly risk of CRC↑ ↑ risk of CRC [53] Lohsoonthorn [53]et al. Lohsoonthorn 1995 279 et al. 1995 [40] Rosato 279 279 et al. 2013 279 329 Bacon 1361 Bacon 12.49 Processed (1.68–269.1) meat 12.49 (1.68–269.1) Significantly ↑ 1.56 bacon (1.11–2.20) Significantly consumption ↑ bacon in cases consumption group in cases Significantly group risk of CRC [62] Dales et al. 1978 99 Nutrients 280 [63]2019 Joshi, 11 et [41],al. Nitrite 2673 2015 Hu treated et al. 2008 meats 3350 3174 3504 1.22 5039 Processed meats Processed No meats significant risk of 1.20CRC (1.0–1.4) 1.50 (1.2–1.8) No significant Significantly risk of CRC↑ ↑ risk of CRC 7 of 17 [34] Tajima and Tomina[34] Tajima 1985 and Tomina 93 1985 [41] Hu 93 186 et al. 2008 186 Ham 3174 and sausage Ham 5039 and sausage Processed 2.87 meats 2.87 Significantly 1.50 (1.2–1.8) ↑ risk Significantly of CRC ↑ risk of CRCSignificantly risk of CRC [63] Joshi et al. 2015 3350 3504 [64] Balder et al. Processed 2006 meats 1535 4371 1.20 (1.0–1.4) Processed meats No significant risk of 1.18 CRC (0.84–1.64) No significant risk of CRC [36] Haenszel et al. [36]1973 Haenszel et al. 179 1973 [60][60] Williams 179 Williams 357 et etal. al. 2010 2010 357 Sausage and 945 other 945 Sausageprocessed and 959 pork 959 other processed pork Processed 2.3 ProcessedϞ 1.77 Ф meat 2.7 meat҂ 2.3Ϟ 1.77 Ф 2.7 1.36҂ 1.36 (0.80–1.68) (0.80–1.68)SignificantlyՒ 1.02Ւ 1.02↑ (0.38–1.96) risk (0.38–1.96)Significantly of CRCⱦ ⱦ ↑ risk of CRC No No significant significant risk risk of ofCRC CRC [64] Balder et al. 2006 1535 4371 [65] Murtaugh Processed et al. 2003 meats 952 1.18 1205 (0.84–1.64) Processed meat No significant1.18 risk(0.87–1.61) of CRC ♀ 1.23 (0.84–1.81) ♂ No significant risk of CRC [35] Levi et al. 2004[35] Levi et al. 2004 323 [61][61] Benito 323 Benito 1271 et etal. al. 1990 1990 1271 Ham 286 salami 286 sausage Ham 498 498salami sausage Processed 2.53 Processed (1.50–4.27) meat meat 2.53 (1.50–4.27) Significantly 1.36 1.36 ↑ risk Significantly of CRC ↑ risk of CRC No No significant significant risk risk of ofCRC CRC [65] Murtaugh et al. 2003 952 1205 Processed meat ♀ ♂ No significant risk of CRC [38] Bidoli et al. 1992[38] Bidoli et al. 123¥ 1992 125 [66]₸ Kimura[42] 123¥[42] et De al. De125 699 Verdier2007 Verdier₸ et etal. Tableal. 1991 1991 699 3. 782Characteristics 1.18 Salami 559(0.87–1.61) 559 and 793sausages of 1.23 Salami case 505 (0.84–1.81) 505 and control sausages Processed human meats studies 1.8¥Sausage Sausage 1.9₸ assessing 1.8¥ colorectal 1.9₸ 1.15 1.30 (0.83–1.60) 1.30 cancer (0.8–1.9)¥ (0.8–1.9)¥Sig and↑ 1.7¥ 1.7 but(1.1–2.8) processed (1.1–2.8) not ₸Sig₸ ₸ ↑ meat¥ but consumptionnot No ₸ significant SignificantlySignificantly risk of CRC↑ ↑ risk risk of ofCRC CRC [66] Kimura et al. 2007 [37] Young and 782 Wolf [37] 1988 Young and 793 152 Wolf ⱡ 201‡ [68]1988 Nothlings [62][62] 152 Dales Processed etⱡ Dales201‡ 618al. et2009 etal. al. meats1978 1978 618 1009 Processed 99 99 lunch 1.15 1522 (0.83–1.60) meat Processed 280 280 lunch meat Processed Nitrite Nitrite 1.85meat treated(1.33–2.58) treated No meats significant meats 1.85 (1.33–2.58) risk of 1.08 CRC (0.89–1.39) Significantly 1.22 1.22 ↑ risk Significantly of CRC ↑ risk No of significantCRC No No significant risksignificant of CRC risk risk of ofCRC CRC [68] Nothlings et al. 2009 [42] De Verdier 1009 et al. [42] 1991 De Verdier 1522 et 559al. 1991[69] SteinmetzAuthor[63][63] Joshi 559Processedand Joshi 505 etPotter etal. al. 2015 meat 19932015 Cases 505 220 (n =) 3350 3350 Controls Bacon 1.08 438 (0.89–1.39) (n 3504=) 3504 Bacon Description Processed 1.3 (0.8–1.9)¥ of Processed meat Processed 1.7 Nomeats (1.1–2.8) meats 1.3 significant Meat (0.8–1.9)¥ ₸ 1.03 risk 1.7(0.55–1.95) (1.1–2.8)of CRC Relative♂ ₸ 0.77 1.20Sig (0.35–1.68) 1.20 Risk (1.0–1.4)↑ (1.0–1.4) ¥ (CI) but♀ not ₸Sig ↑ ¥ but notNo ₸ significant No No significant risksignificant Findings of CRC risk risk of ofCRC CRC [69] Steinmetz and Potter [39]1993 Navarro et 220 al. 2003[39] Navarro 438et al. 2872003[70] Franceschi[64][64] Balder 287 Processed etBalder 566 al. 1997et etal. al. meat2006 2006 566 1225 Cold1.03 1535cuts (0.55–1.95) 1535 and 4154 sausages ♂ Cold 0.77 4371 cuts 4371(0.35–1.68) and sausages♀ Processed Processed 1.64meat Processed (1.16–2.32) Nomeats meats significant 1.64 (1.16–2.32) risk of 1.02 CRC (0.89–1.24) Significantly 1.18 1.18 (0.84–1.64) (0.84–1.64) ↑ risk Significantly of CRC ↑ risk No of significantCRC No No significant risksignificant of CRC risk risk of ofCRC CRC [70] Franceschi et al. 1997 1225 [62] Dales et 4154al. 1978[ 53 ][71] Lohsoonthorn Centozone Processed 992009 et al. 1995 meat 280 279 119 1.02 119 279 (0.89–1.24) Nitrite treated meats Processed Bacon meat No significant 1.22 risk of CRC 12.49 1.01 (1.68–269.1) No significant Significantly risk Noof CRC significant bacon risk consumption of CRC in cases group [62] Dales et al. 1978 99 [65][65] Murtaugh Murtaugh 280 et etal. al. 2003 2003 Nitrite 952 952 treated meats 1205 1205 Processed Processed 1.22 meat meat 1.181.18 (0.87–1.61) (0.87–1.61) No significant ♀ 1.23♀ 1.23 (0.84–1.81)risk (0.84–1.81) of CRC ♂ ♂ ↑NoNo significant significant risk risk of ofCRC CRC [71] Centozone 2009 [43] Macquart-Moulin 119 [43] 1986Macquart-Moulin 119 [54 399][72] De 1986 Tiemersma Stefani [66][66] et Kimura 3 Processed al.99Kimuraet399 al. 2012 2002 et etal. meat al. 2007 2007 399 321 102 782 Charcuterie 782 537 844 1.01 793 Charcuterie 793 Processed Sausage Processed Processed meat 0.89 Nomeats meats significant 0.89 risk 3.53 of 0.90CRC (1.93–6.46) (0.6–1.3) No 1.15 1.15significant 2.01(0.83–1.60) (0.83–1.60) (1.07–3.76) risk Noof CRC significant risk Noof CRC significant Significantly No No significant risksignificant of CRCrisk risk risk ofof of CRC CRC [80] Iscovich et al. 1992 110 220 Delicatessen meat 0.43 (0.21–0.89) ↓ Significantly ↓ risk of CRC ↑ [72] Tiemersma et al. 2002 [80] Iscovich et 102 al. 1992 537 [34110][43] Tajima Macquart-Moulin and[68][68] TominaNothlings Nothlings 220 Sausage 19851986 et et al. al. 2009 2009 93 399 Delicatessen 1009 1009 0.90 399 186 meat (0.6–1.3) 1522 1522 Ham Charcuterie and Processed 0.43 Processed sausage (0.21–0.89) Nomeat meat significant risk of CRC 0.89 Significantly1.08 2.871.08 (0.89–1.39) (0.89–1.39) risk of CRC Significantly Significantly No No ↑significant significantrisk of CRCrisk risk risk ofof of CRC CRC [45] Nowell et al. 2002[45] Nowell et al. 2002 157 157 380 380 Sausage and bacon Sausage and bacon - ↑ - Significantly ↑ bacon Significantly consumption ↑ bacon in cases consumption group in cases↓ group ↑ [43] Macquart-Moulin 1986 399 399 [35][80] Levi Iscovich et al.[69][69] 2004 et Steinmetz al.Steinmetz Charcuterie 1992 and and Potter Potter 1993 1993 323110 220 220 220 1271 0.89 438 438 Processed Processed Processed meat Processed meat Significantly meat meat risk of 0.431.03 1.03CRC (0.21–0.89)(0.55–1.95) 2.53 (0.55–1.95) (1.50–4.27) ♂ ♂0.77 0.77 (0.35–1.68) (0.35–1.68)♀ ♀ Significantly SignificantlyNoNo significant significantrisk of CRCrisk risk risk ofof of CRC CRC ↓ ↑ ↑ [80] Iscovich et al. 1992 ↑ increased;110 ↓↑decreased; increased; 220 [36 ] ⱡ[45] Haenszel↓ Proximaldecreased; Nowell[70][70]et et Franceschicolon; al.al Processed Franceschi. 2002ⱡ 1973 Proximal ‡ distal etmeat etal. al. 1997colon; colon; 1997 CRC 179 ‡ 157 distal colorectal colon; 1225 1225 0.43 CRCcancer, 380 357 (0.21–0.89) colorectal ¥ 4154 colon, 4154 Sausage cancer, ₸ Sausage rectum, and¥ colon, and other Processed♂ bacon Processed male; processed₸ rectum, Significantly♀ meat female; meat pork ♂ male; Ϟ riskHawaiian; ♀ female; of CRC2.30 - Ϟ 1.02Ф 1.77 1.02 Hawaiian; Issei;(0.89–1.24) φ(0.89–1.24)2.7 ҂ Nisei. Ф SignificantlyIssei; Where ҂ Nisei. baconSignificantly Where No consumption No significant significantrisk riskin risk cases ofof of CRC groupCRC ↑ ↑ [45] Nowell et al. 2002 the authors 157 did thenot authors provide 380 [37 did the] Young notrelative↑ provide andincreased;[71][71] risks Sausage WolfCentozone Centozonethe for 1988 relative ↓ andmales decreased2009 bacon 2009 and risks 152female for ⱡ males201 Proximals combined, and 119 119 female colon; we618 s‡have combined, -distal 119 provided 119 colon; we ♂ the Processedhave male; gender provided Significantly♀ Processedfemale; lunch Processedspecific the meat¥ meat colon;genderrelative meat bacon specific₸ riconsumption sks. rectal; Relative relative Ւ in1.85 caucasion;casesrisk risks. (1.33–2.58) values group Relative 1.01 1.01 reflectⱦ African risk those values American; in thereflect thoseCRC Significantly colorectalin No the No significant significant cancer.risk risk risk Whereofof of CRC CRC ‡ ↑ ↑ increased; ↓ decreased ⱡ Proximalhighest colon; consumption[ ‡38 distal] Bidoli thecolon; et groupauthors[72] al.[72] ♂ Tiemersma 1992 male;Tiemersma vs did those ♀ not female; et in provideetal. al. the2002 2002¥ 123¥ lowest colon; the 125 relative consumption ₸ rectal; 102 risks 102 forՒ699 grou malescaucasion;p. 537 All 537and relative female ⱦ African Salami srisk combined, values andAmerican; Sausagesausages are Sausagewe adjusted,have CRC provided colorectal with the the cancer. exceptiongender Where1.8¥ specific 0.90 0.90o 1.9f (0.6–1.3) Iscovich (0.6–1.3) relative et rialsks. and Relative Nowell risk et al. values more No NoSig significant reflect significant¥ but those risk not risk ofin ofCRC the CRC highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al and Nowell et al. more ↑ the authors did not provide the relative risks for[ 39males] Navarro and female[43] et[43] al.Macquart-Moulin Macquart-Moulins 2003 combined, we 1986 1986have 287 provided 399 399 the gender 566 specific 399 399 relative Cold cutsrisks. and CharcuterieRelative Charcuterie sausages risk values reflect those 1.64 in (1.16–2.32) the 0.89 0.89 Significantly Significantly Significantly ↑ risk↑ risk risk of ofCRC ofCRC CRC details of adjustmentsdetails areof adjustments providedhighest in aresupplementary providedconsumption in supplementarytables group 1 andvs those 2. tables in the 1 andlowest 2. consumption group. All relative risk values are adjusted, with the exception of Iscovich et al and Nowell↑ et al. more [40] Rosato et[80][80] al. Iscovich 2013 Iscovich et etal. al. 1992 1992 329110110 1361 220 220 Processed Processed Processed meat meat meat 1.56 0.43 (1.11–2.20) 0.43 (0.21–0.89) (0.21–0.89) Significantly Significantly Significantly ↓ risk↓ risk risk of ofCRC ofCRC CRC highest consumption group vs those in the lowest consumptiondetails of grouadjustmentsp. All relative are prov riskided values in supplementary are adjusted, withtable the 2. exception of Iscovich et al and Nowell et al. more ↑ [41] Hu et al.[45] 2008[45] Nowell Nowell et etal .al 2002. 2002 3174 157 157 5039 380 380 Processed Sausage Sausage meats and and bacon bacon 1.50 (1.2–1.8) - - Significantly Significantly Significantly ↑ ↑ bacon bacon consumption consumptionrisk of CRC in incases cases group group details of adjustments are provided in supplementary table 2. ↑ [60] Williams et al.↑↑ 2010increased; increased; ↓ ↓ decreased decreased 945 ⱡ ⱡ Proximal Proximal 959 colon; colon; ‡ distal‡ distal colon; colon; Processed ♂ ♂male; male; meat ♀ female;♀ female; ¥ colon;¥ colon;1.36 ₸ (0.80–1.68) ₸ rectal; rectal; Ւ Ւ1.02 caucasion; caucasion; (0.38–1.96) ⱦ ⱦ African African American; American;No significantCRC CRC colorectal colorectal riskof cancer. CRCcancer. Where Where [61] Benito et al.the 1990the authors authors did did not not provide 286 provide the the relative relative 498 risks risks for for males males and and Processedfemale females combined,s meatcombined, we we have have provided provided the the gender 1.36 gender specific specific relative relative ri sks.risks. Relative Relative No significantrisk risk values values risk reflect reflect of CRC those those in in the the [42] De Verdier et al. 1991 559 505 Sausage 1.30 (0.8–1.9)¥ 1.7 (1.1–2.8) Significantly risk of CRC highesthighest consumption consumption group group vs vs those those in in the the lowest lowest consumption consumption grou group.p. All All relative relative risk risk values values are are adjusted, adjusted, with with the the exception exception o fo Iscovichf Iscovich et etal aland and↑ Nowell Nowell et etal. al. more more [62] Dales et al. 1978 99 280 Nitrite treated meats 1.22 No significant risk of CRC detailsdetails of of adjustments adjustments are are prov providedided in in supplementary supplementary table table 2. 2. [63] Joshi et al. 2015 3350 3504 Processed meats 1.20 (1.0–1.4) No significant risk of CRC [64] Balder et al. 2006 1535 4371 Processed meats 1.18 (0.84–1.64) No significant risk of CRC [65] Murtaugh et al. 2003 952 1205 Processed meat 1.18 (0.87–1.61) 1.23 (0.84–1.81) No significant risk of CRC [66] Kimura et al. 2007 782 793 Processed meats 1.15 (0.83–1.60)♀ ♂ No significant risk of CRC [68] Nothlings et al. 2009 1009 1522 Processed meat 1.08 (0.89–1.39) No significant risk of CRC [69] Steinmetz and Potter 1993 220 438 Processed meat 1.03 (0.55–1.95) 0.77 (0.35–1.68) No significant risk of CRC [70] Franceschi et al. 1997 1225 4154 Processed meat 1.02 (0.89–1.24)♂ ♀ No significant risk of CRC [71] Centozone 2009 119 119 Processed meat 1.01 No significant risk of CRC [72] Tiemersma et al. 2002 102 537 Sausage 0.90 (0.6–1.3) No significant risk of CRC [43] Macquart-Moulin 1986 399 399 Charcuterie 0.89 Significantly risk of CRC ↑ [80] Iscovich et al. 1992 110 220 Processed meat 0.43 (0.21–0.89) Significantly risk of CRC ↓ [45] Nowell et al. 2002 157 380 Sausage and bacon - Significantly bacon consumption in cases group ↑ increased; decreased Proximal colon; distal colon; male; female; ¥ colon; rectal; caucasion; African American; CRC colorectal cancer. Where the authors did not provide the ↑ ↓ ‡ relative risks for males and females combined, we have provided♂ ♀ the gender specific relative risks. Relative risk values reflect those in the highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al. and Nowell et al. more details of adjustments are provided in Supplementary Table S2. Nutrients 2019, 11, 2673 8 of 17

Although an equal number of studies prove/disprove the link between processed meat and CRC, those studies which found a relationship generally involved more participants; this explains why numerous meta-analyses have concluded that processed meat is a risk factor for CRC [5–7]. One study reported no association between processed meat consumption and CRC in men (n = 241), but reported a beneficial association between processed meat and CRC in women (n = 197) [69]. Two further prospective studies found that processed meat consumption was associated with CRC in men but not in women [37,59]. These findings were supported by three further studies (n = 119,260) indicating no effect of processed meat in female cases and controls [32,55,79]. A study of almost 48,000 American men reported that the association between processed meat consumption and CRC did not reach significance (p = 0.06) [73].

Studies Focusing on Nitrite-Containing Meat and CRC Of the human studies discussed above, 17 studies investigated nitrite-containing processed meats (Table4). Five studies found nitrite-containing processed meats to have no e ffect on CRC [43,56,57,59,62] and one study found nitrite-containing processed meat was protective [80]. In contrast, a total of 11 studies found that nitrite-containing processed meat increases the risk of CRC [34–39,42,45,48,49,53]. This indicates that the proposed causal relationship between processed meat is potentially skewed by the intake of nitrite-containing processed meats. Of the prospective studies discussed (n = 5), three used the International Classification of Diseases criteria to confirm that the participant met the conditions of a positive diagnosis of CRC [49,57,59]; two studies did not specify which criteria they used. Wu et al. [48] consulted the patients’ medical records. Takachi et al. [56] conducted a linkage study, where the researchers had access to a local cancer registry. Of the case-control studies (n = 12), ten studies recruited participants that had histologically confirmed adenocarcinoma; only one stated that the International Classification of Diseases criteria was applied to their study population [80]. Dales et al. [62] recruited hospitalized CRC patients, whilst Young and Wolf [37] recruited participants from the Wisconsin Cancer Reporting System. All studies used a food frequency questionnaire (FFQ) to capture processed meat consumption in their respective cohorts; only two of the studies set out with the intention of investigating the role of nitrite in CRC development [59,62] and both of these found no relationship. No study considered the concomitant consumption of haem proteins, and only one study considered the effect of nitrosamine exposure [59] levels; however, no study directly measured that. The FFQs varied in design and detail. Three studies recorded information on portion size [43,45,57], and the remainder did not. Researchers invited participants to describe their consumption habits in a number of ways, Dales et al. [62] included eight different frequencies, ranging from “never” to “at least once a day.” Four studies provided their participants with six frequencies to choose from [34,37,48,80]. Two studies used five categories of frequency [42,57], Lohsoonthorn et al. [53] used four categories; the remainder of the studies did not specify how many categories were available (n = 10). Only one study described their FFQ as validated [39]. It has been shown that illness substantially effects dietary intake, and it is, therefore, crucial that all studies investigating the causative effect of habitual dietary pattern, consider the period prior to illness. Of the studies that recruited participants with existing CRC (n = 12), seven studies stated that participants were instructed to record dietary information on periods prior to illness. These periods ranged from weekly to over the course of the participant’s lifetime. Nutrients 2019, 11, x FOR PEER REVIEW 7 of 17 Nutrients 2019, 11, x FOR PEER REVIEW 7 of 17 Table 3. Characteristics of case control human studies assessing colorectal cancer and processed meat consumption Table 3. Characteristics of case control human studies assessing colorectal cancer and processed meat consumption Author Cases (n =) Controls (n =) Description of processed meat Relative Risk (CI) Findings ↑ [53] Lohsoonthorn et al. 1995Nutrients 2019 279, 11Nutrients, x FOR PEER 2019 279, REVIEW11, x FOR PEER AuthorREVIEW Bacon Cases (n =) Controls 12.49 (1.68–269.1) (n =) Description10 of of Significantly processed 17 meat 10 of bacon 17 consumption Relative in cases Risk group(CI) Findings [54] De Stefani et al. 2012 321 844 [53] Lohsoonthorn Processed et al. 1995 meat 279 3.53 (1.93–6.46) 279 2.01 (1.07–3.76) Bacon Significantly ↑ risk 12.49of CRC (1.68–269.1) Significantly ↑ bacon consumption in cases group [34] Tajima and Tomina 1985 93 186 [54] De Stefani Ham et al. and 2012 sausage 321 844 2.87 Processed meat Significantly ↑ 3.53 risk (1.93–6.46) of CRC 2.01 (1.07–3.76) Significantly ↑ risk of CRC ↑ [35] Levi et al. 2004 323 1271Table [34] 4. TajimaCharacteristics andTable Processed Tomina 4. Characteristicsof 1985meatprospective andof 93 prospective case control 2.53 186and human (1.50–4.27) case controlstudies humanassessing Ham and studies colorectalsausage assessing Significantly cancer colorectal and nitrite risk cancer of containing CRC and 2.87 nitrite meat containing meat Significantly ↑ risk of CRC [36] Haenszel et al. 1973 179 357 [35] Levi Sausage et al. and 2004 other processed pork 323 2.30 1271Ϟ 1.77Ф 2.7҂ Processed meat Significantly ↑ risk of 2.53 CRC (1.50–4.27) Significantly ↑ risk of CRC [37] Young and Wolf 1988 152 Authorⱡ 201 ‡ 618Author Sample [36] size Haenszel Colorectal Processed Sample et al. 1973 sizecancer lunch Colorectal casesmeat cancer 179 Description cases 1.85 of 357 processed (1.33–2.58) Description meat Sausage of processed and other meat processed Relative Significantly porkRisk (CI) Relative ↑ risk Risk of 2.30 CRC(CI)Ϟ 1.77 Ф 2.7҂ Findings FindingsSignificantly ↑ risk of CRC Nutrients 2019, 11, 2673 9 of 17 [38] Bidoli et al. 1992 [48] Wu 123¥ et al. 125 2006₸ [48] Wu et 699al. 2006 14,032 [37] Young and Salami 14,032Wolf and 581 1988 ‡ sausages 152581 ‡ⱡ 201 ‡ Sausage, salami, 618 1.8¥ 1.9bologna Sausage,₸ salami, Processed bologna lunch 1.52meat (1.12–2.08) Sig ↑ ¥ 1.52 but (1.12–2.08) not 1.85 ₸ (1.33–2.58) Significantly ↑ risk Significantly of CRC ↑ Significantly risk of CRC ↑ risk of CRC Salami, continentalSalami, sausages, continental sausages sausages, or sausages or ↑ [39] Navarro et al. 2003 287 [49] English 566 et al. 2004[38] Bidoli Coldet al. 37,1121992cuts and sausages 123¥ 284 125₸ 1.64 699 (1.16–2.32) Salami and sausagesSignificantly 1.50 risk(1.1–2.0) of CRC 1.8¥ 1.9₸ ↑ Significantly ↑ riskSig of CRC↑ ¥ but not ₸ [49] English et al. 2004 37,112 284 frankfurters, bacon, ham 1.50 (1.1–2.0) Significantly risk of CRC [40] Rosato et al. 2013 329 1361 [39] Navarro et Processed al. 2003 meat 287 frankfurters, 1.56 566 bacon,(1.11–2.20) ham Cold cuts and sausages Significantly ↑ risk of 1.64 CRC (1.16–2.32) Significantly ↑ risk of CRC [56] Takachi et al. 2011 98,514 1145 Processed meat 1.27 (0.95,1.71) ♂ 1.19 (0.82,1.74) ♀ No significant risk of CRC [41] Hu et al. 2008 [56] Takachi 3174 et al. 2011 5039 98,514[40] Rosato et Processedal. 2013 1145 meats 329 Processed 13611.50 (1.2–1.8) meat Processed1.27 (0.95,1.71) meat Significantly ♂ 1.19 (0.82,1.74) ↑ risk♀ of 1.56 CRC (1.11–2.20) No significant risk of CRC Significantly ↑ risk of CRC [57] Sato et al. 2006 47,605Table 4. Characteristics 358 of prospective Ham andor sausage case control human studies 0.91 (0.61–1.35) assessing colorectal cancer No significant and nitrite risk of containing CRC meat [60] Williams et al. 2010 [57] Sato et 945 al. 2006 959 47,605[41] Hu et al. 2008 Processed 358 meat 3174 1.36 (0.80–1.68) Ham 5039or sausage Ւ 1.02 (0.38–1.96)ⱦ Processed meats 0.91 (0.61–1.35) No significant risk of CRC 1.50 (1.2–1.8) No significant risk of CRC Significantly ↑ risk of CRC [59] Knekt et al. 1999[59] Knekt et al. 1999 9,985 9,985 73 73 Nitrite Nitrite 0.74 (0.34–1.63) 0.74 (0.34–1.63) No significant risk No of CRCsignificant risk of CRC [61] Benito et al. 1990 286 498 [60] Williams et Processed al. 2010 meat 945 959 1.36 Processed meat No significant 1.36 risk (0.80–1.68) of CRC Ւ 1.02 (0.38–1.96)ⱦ No significant risk of CRC Author Author Cases (n =) CasesControls (n =) (n =) Controls (n Description=) Colorectal of processed Description meat of processed meat Relative Risk (CI) Relative Risk (CI) Findings Findings [42] De Verdier et al. 1991 559 505 [61] BenitoAuthor et al. 1990 Sausage Sample 286 size 1.30 (0.8–1.9)¥ 498 1.7 (1.1–2.8)₸Description Processed meat of Processed Significantly Meat ↑ risk of CRC 1.36 Relative Risk (CI) No significant risk Findingsof CRC [53] Lohsoonthorn[53] et al. Lohsoonthorn 1995 279et al. 1995 279 279 279 Cancer Bacon Cases Bacon 12.49 (1.68–269.1) 12.49 (1.68–269.1) Significantly ↑ bacon Significantly consumption ↑ bacon in cases consumption group in cases group [62] Dales et al. 1978 99 280 [42] De Verdier Nitrite et al. treated 1991 meats 559 505 1.22 Sausage No significant 1.30risk (0.8–1.9)¥of CRC 1.7 (1.1–2.8)₸ Significantly ↑ risk of CRC [34] Tajima and Tomina[34] Tajima 1985 and Tomina 93 1985 93 186 186 Ham and sausage Ham and sausage 2.87 2.87 Significantly ↑ risk Significantly of CRC ↑ risk of CRC [63] Joshi et al. 2015 3350 3504[48 ] Wu[62] etDales al. 2006et al. Processed 1978 meats 14,032 99 1.20 280 581 (1.0–1.4) NitriteSausage, treated salami, meats Nobologna significant risk of CRC 1.22 1.52 (1.12–2.08) No significant Significantly risk of CRCrisk of CRC [36] Haenszel et al.[36] 1973 Haenszel et al. 1791973 179 357 357 Sausage and other Sausage processed‡ and pork other processed pork 2.3Ϟ 1.77 Ф 2.7҂ 2.3Ϟ 1.77 Ф 2.7҂ Significantly ↑ riskSignificantly of CRC ↑ risk of CRC ↑ [64] Balder et al. 2006 1535 4371 [63] Joshi et al. Processed 2015 meats 3350 1.18 3504 (0.84–1.64) Salami, Processed continental meats No sausages, significant risk of CRC 1.20 (1.0–1.4) No significant risk of CRC [35] Levi et al. 2004[35] Levi et al.[49 2004] English 323 et al. 2004 323 1271 37,112 1271 Ham salami 284 sausage Ham salami sausage 2.53 (1.50–4.27) 2.53 (1.50–4.27) 1.50 Significantly (1.1–2.0) ↑ risk Significantly of CRC ↑ risk of CRC Significantly risk of CRC [65] Murtaugh et al. 2003 952 1205 Processed meat 1.18 (0.87–1.61) ♀ 1.23 (0.84–1.81)sausages ♂ or frankfurters,No bacon,significant ham risk of CRC [38] Bidoli et al. 1992[38] Bidoli et al. 123¥1992[64] 125 Balder₸ et al. 123¥ 2006 125 699 ₸ 699 1535 Salami and 4371 sausages Salami and sausages Processed meats 1.8¥ 1.9₸ 1.8¥ 1.9 1.18₸ (0.84–1.64)Sig ↑¥ but not ₸Sig ↑¥ but No not significant ₸ risk of CRC↑ [66] Kimura et al. 2007 [37] Young 782 and Wolf[37] 1988 Young 793 and[56 152Wolf] Takachi[65]ⱡ 201‡ 1988 Murtaugh et al. Processed 152 2011et al.ⱡ 201‡ 6182003 meats 98,514 618 952 Processed 1.15 1205 1145lunch (0.83–1.60) meat Processed lunch Processed meat Processed meat 1.85 meat(1.33–2.58) No significant 1.18 1.85 risk (1.33–2.58)(0.87–1.61) of 1.27 CRC (0.95,1.71) ♀ 1.23 Significantly (0.84–1.81)1.19 (0.82,1.74)↑♂ risk Significantly of CRC ↑ No risk significantof CRCNo significant risk of CRC risk of CRC [68] Nothlings et al. 2009 [42] De Verdier 1009 et [42]al. 1991 De Verdier 1522[57 et] Satoal.[66] 559 1991 Kimura et al. 2006 et Processedal. 5592007 505 meat 47,605 505 782 1.08 Bacon 793 358(0.89–1.39) Bacon Processed Ham 1.3 meats (0.8–1.9)¥ or sausage No 1.7 significant (1.1–2.8) 1.3 (0.8–1.9)¥₸ risk 1.7 of 1.15 (1.1–2.8)CRC (0.83–1.60) ₸ 0.91 (0.61–1.35)Sig ♂ ↑ ¥ but not Sig₸ ♀↑ ¥ but No not significant ₸ No significant risk of CRC risk of CRC [69] Steinmetz and Potter 1993[39] Navarro 220 et al. [39]2003 Navarro 438[ et59 al.] Knekt2003[68] 287 Nothlings et al. 1999 Processedet 287al. 2009 566 meat 5669985 1009 1.03 Cold (0.55–1.95) cuts 1522 and 73 ♂sausages 0.77 Cold (0.35–1.68) cuts and sausages♀ Processed Nitritemeat 1.64 (1.16–2.32)No significant 1.64 risk (1.16–2.32) of 1.08 CRC (0.89–1.39) 0.74 Significantly (0.34–1.63) ↑ risk Significantly of CRC ↑ Norisk significantof CRC No significant risk of CRC risk of CRC [70] Franceschi et al. 1997 1225 [62] Dales 4154 et al. 1978 Processed 99 meat 280 1.02 (0.89–1.24) Nitrite treated meats No significant risk 1.22 of CRC No significant risk of CRC [62] Dales et al. 1978 [69] 99 SteinmetzAuthor and Potter 280 1993 Cases 220 (n =) Nitrite Controls treated 438 meats (n =) Description Processed meat of Processed 1.22 Meat1.03 (0.55–1.95)♂ Relative 0.77 No (0.35–1.68) significant Risk♀ (CI) risk of CRC No significant risk Findingsof CRC [71] Centozone 2009 [43] Macquart-Moulin 119 [43] Macquart-Moulin1986 119 [70] 399 1986Franceschi Processed et 3 al.99 3991997 meat 399 1225 Charcuterie 4154 1.01 Charcuterie Processed meat 0.89 No significant risk 0.89 of 1.02 CRC (0.89–1.24) No significant risk No of CRCsignificant risk No of significant CRC risk of CRC [72] Tiemersma et al. 2002 [80] Iscovich 102 et al. [80]1992 Iscovich 537 [et53 al.] Lohsoonthorn1992[71]110 Centozone 2009 et110 Sausage al.220 1995 220 279 119 Delicatessen 0.90 119 279 (0.6–1.3) meat Delicatessen Processedmeat meat Bacon 0.43 (0.21–0.89) No significant 0.43 risk (0.21–0.89) of CRC 1.01 12.49 Significantly (1.68–269.1) ↓ risk Significantly of CRC Significantly↓ Norisk significantof CRC bacon risk of consumption CRC in cases group ↑ ↑ [43] Macquart-Moulin 1986 [45] Nowell 399 et al. 2002[45] Nowell 399 et[34 al.] 2002 Tajima[72] 157 Tiemersma and Tomina et Charcuterie 157 al. 380 2002 1985 380 93 102 Sausage 537 and 186 0.89 bacon Sausage and bacon Sausage Ham and sausage Significantly - ↑ risk - of Significantly 0.90 CRC (0.6–1.3) ↑ 2.87 bacon Significantly consumption bacon in cases consumption group No significant in Significantly cases risk group of CRC risk of CRC ↑ [80] Iscovich et al. 1992 ↑ increased;110 ↓↑decreased; increased; 220[36 ] Haenszel[43] ⱡ↓ Macquart-Moulin decreased; Proximal et al. Processed 1973colon; ⱡ 1986 Proximal ‡meat distal colon;colon; 179 399 CRC‡ distal colorectal colon; 0.43 399 CRC 357cancer,(0.21–0.89) colorectal ¥ colon, Sausage cancer, ₸ Charcuterie rectum, and¥ colon, other ♂ male; processed₸ Significantly rectum, ♀ female; pork ♂ ↓male; Ϟrisk Hawaiian; ♀of female; CRC 0.89 2.3 ϞФ 1.77 Hawaiian; Issei;φ 2.7 ҂ Nisei.Ф Issei; Where ҂ Significantly Nisei. SignificantlyWhere ↑ risk of CRCrisk of CRC ↑ ↑ [45] Nowell et al. 2002 the authors 157 didthe not authors 380provide[35 ] Levi [80]did the Iscovich etnot relative al. provide 2004 Sausageet risksal. 1992 the forand relative malesbacon risksand female 323for110 males s combined, and female 220 1271 we -s havecombined, provided we Processed havethe Ham Significantlygender provided salami meat specific sausage the bacongenderrelative consumption specific risks. 0.43Relative relative in (0.21–0.89) cases 2.53 riskri sks.group (1.50–4.27) values Relative reflect risk those values in reflectthe Significantly those Significantly in ↓ the risk of CRCrisk of CRC ↑ ↑ increased; ↓ decreased ⱡ Proximalhighest colon;[38 consumption‡ ]distal Bidoli[45] Nowell colon; et al. group et♂ 1992 almale;. 2002 vs ♀ those female; in the¥123¥ colon; lowest 125157 ₸ consumption rectal; 380Ւ699 caucasion;group. All relative ⱦ Sausage African Salami risk and American; values andbacon sausages are CRC adjusted, colorectal with cancer.the exception -Where 1.8¥ of 1.9 Iscovich et Significantlyal and Nowell ↑ bacon et al. consumptionmoreSig ¥ but in notcases group highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al and Nowell et al. more ↑ the authors did not provide the relative risks for[37 males] Young and↑ and femaleincreased; Wolfs combined, 1988 ↓ decreased we 152have ⱡ 201 provided Proximal the colon; gender618 ‡ distal specific colon; relative ♂ male; Processed risks. ♀ female; Relative lunch ¥ colon; meatrisk values ₸ rectal;reflect thoseՒ caucasion; in 1.85 the (1.33–2.58) ⱦ African American; CRC colorectal Significantly cancer.risk Where of CRC details of adjustmentsdetails ofare adjustments provided in are supplementary provided in supplementary tables 1 and‡ 2. tables 1 and 2. ↑ [42] De Verdierthe authors et al. 1991did not provide the 559 relative risks for 505 males and females combined, Bacon we have provided the gender1.3 (0.8–1.9)¥ specific 1.7 relative (1.1–2.8) risks. Relative risk values reflectSig ¥ those but not in the highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al and Nowell et al. more ↑ [39] Navarro et al. 2003 287 566 Cold cuts and sausages 1.64 (1.16–2.32) Significantly risk of CRC details of adjustments are provided in supplementary tablehighest 2. consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al and Nowell et↑ al. more [62] Dales et al. 1978 99 280 Nitrite treated meats 1.22 No significant risk of CRC details of adjustments are provided in supplementary table 2. [43] Macquart-Moulin 1986 399 399 Charcuterie 0.89 No significant risk of CRC [80] Iscovich et al. 1992 110 220 Delicatessen meat 0.43 (0.21–0.89) Significantly risk of CRC ↓ [45] Nowell et al. 2002 157 380 Sausage and bacon - Significantly bacon consumption in cases group ↑ increased; decreased; Proximal colon; distal colon; CRC colorectal cancer, ¥ colon, rectum, male; female; Hawaiian; φ Issei; Nisei. Where the authors did not provide the ↑ ↓ ‡ relative risks for males and females combined, we have provided the gender specific relative risks. Relative♂ risk♀ values reflect those in the highest consumption group vs those in the lowest consumption group. All relative risk values are adjusted, with the exception of Iscovich et al. and Nowell et al. more details of adjustments are provided in Supplementary Tables S1 and S2. Nutrients 2019, 11, 2673 10 of 17

The above analysis of prior studies clearly indicates that there is a need for methodical studies which specifically investigate nitrite exposure, and which control for confounding factors, such as haem, and saturated fat intake. As a pre-requisite, a well-designed, validated FFQ focusing on processed meat must include portion size. An early report concluded that although processed meat consumption was linked to CRC development [36], nitrite was not responsible. This conclusion was based on the finding that meat with no nitrite had a higher relative risk than meat with nitrite [36]. The authors postulated that meat consumption was acting as a surrogate measure of saturated fat consumption, and that in fact, saturated fat intake could be responsible for the positive association [36]. Further investigation found that the frequency of consumption of nitrite-containing foods and high fat containing foods were not different between cases or controls [62]. This hypothesis is disputed by a study that found controlling for meat intake substantially decreased the association between fat intake and CRC development, suggesting that meat intake was responsible for the relationship [81]. Positive relationships between CRC and the consumption of salami [38], sausages [39], ham [34], and bacon [45] have been reported, although Sato et al. [57] found no relationship between CRC and sausage or ham intake. The content of a sausage differs greatly depending on the location it is being manufactured in. Sausages made in continental Europe tend to contain sodium nitrite, whilst British/Irish sausages do not. As the aforementioned studies that measured sausage intake were conducted in Argentina and Japan, respectively, it is difficult to determine the proportion of nitrite-containing sausages that the populations actually consumed. In a prospective study of 9985 participants, it was reported that N-nitrosodimethylamine consumption was positively related to CRC development; when locating the origin of the N-nitrosodimethylamine, there was a strong significant association between intake of smoked and salted fish and risk of CRC; however, the association between intake of cured meat and sausages with CRC was not significant [59]. A case-control study found that CRC patients were more likely to consume processed lunchmeat at various stages of their life compared with controls, and furthermore, it was speculated that additives such as nitrites may be responsible for this association [37]. Pierre et al. (2013) [22] conducted the only human intervention study in this area. Seventeen males were asked to abstain from meat and antioxidants for a 7-day control period, following which they consumed 180 g of ham per day for 4 days; after a washout period, the same participants consumed 180 g of tocopherol enriched ham a day for 4 days, and finally, 180 g of ham and 500 mg of calcium per day. During the ham-only period, participants had significantly higher faecal levels of apparent total N-nitroso compounds (ATNC) and thiobarbituric acid reactive substances (TBARS) than during the control periods. The addition of calcium to the diet mitigated this rise. There was no change in the faecal water cytotoxicity or 1,4-Dihydroxynonane mercapturic acid (DHN-MA) following any treatment. None of the markers measured in that study are validated markers of colorectal cancer; however, they are the best available without imaging or visualising the colon. The existing human evidence indicates that nitrites, through their exogenous and endogenous conversion to NOCs, are an important contributing factor in the proposed causal link between processed meat consumption and CRC.

4. Potential Mechanisms Up to 75% of NOC exposure can be attributed to endogenous production [82]. NOCs are formed following the ingestion of nitrite. The mechanism responsible for this is the electron exchange reaction involving amines and nitrogen oxides; this occurs most efficiently when at low pH [83]. NOCs are also encountered from exogenous sources, such as cigarette smoking and the diet. Nitrosamines are also exogenously produced, commonly occurring in foods such as cheese, fish, beer, and water. Common forms of exogenous derived nitrosamines are N-nitrosodimethyamine (NDMA); N-nitrosodiethylamine (NDEA); Nnitrosodibutylamine (NDBA); and N-nitrosopiperidine (NPIP). Not all of these compounds are carcinogens but some have been classified as probable by IARC [84]. The enzymatic incorporation of a hydroxyl group to N-nitrosamines catalysed by cytochrome P450 2E1 (CYP2E1) yields diazomethane, and ultimately, DNA-reactive methyl carbocation [85]. Nutrients 2019, 11, 2673 11 of 17

This involves the formation of methylated DNA adducts, by the addition of an alkyl group to oxygen within DNA bases, which leads to the production of two mutagenic nucleobases, O6–methylguanine and O4–methylthymine [86]. The relationship between CYP2E1 and NOC metabolism is so profound that it has been reported that depletion of CYP2E1 confers resistance to smoking-induced lung cancer [87]. Haem centres can reduce nitrite to nitric oxide (NO), and depending on substrate availability, nitrosation can then occur. It has been shown that the nitrosation of amines can lead to the 6 production of diazoacetate and ultimately O -carboxymethyl-20-deoxy-guanosine, which is a further NOC DNA adduct [88]. Multiple nutrients have been shown to prevent the formation of NOCs, including vitamin C, vitamin E, and chlorophyll. It is well established that vegetables contain signiﬁcant amounts of nitrates which can be reduced by bacteria during mastication to form nitrite, and this nitrite enters the stomach for nitrosation. However, as vegetables do not contain haem, instead containing antioxidants such as vitamin C, vitamin E, and chlorophyll, the nitrite derived from vegetables does not pose the same mutagenic risk as processed meat.

5. Discussion CRC is a somatic genetic disease and its onset is influenced by both the colonic environment and the patient’s genetic background. Increased CRC risk is evident in populations of particular dietary patterns. Studies that have focused on singular foodstuffs or nutrients have frequently yielded inconclusive findings, indicating that the risk of CRC is altered depending on dietary pattern rather than simply the consumption of a particular food. Thus, by far the majority of studies have not considered the cumulative effects of multiple risk factors, including increased abdominal adipose tissue, cigarette smoking, physical inactivity, alcohol intake, consumption of saturated fats, and low fibre consumption. The long-term, habitual consumption of processed meat is likely to be another contributing factor [89,90]. It is evident from the pre-clinical studies that haem is a promotor of CRC development; however, it is unclear from the human evidence if it is simply a confounding factor or an important contributor. The majority of human studies conducted have included multivariate analysis to control for confounding factors; however, too frequently the researchers rely on self-reported data. It is clear from the proposed mechanism that nitrite requires other nutrients to be present to produce carcinogenic compounds. Future research should take a holistic dietary approach when analysing exposure data. The majority of nitrates ingested in the diet come from vegetable consumption. As previously stated, nitrates are reduced during mastication to form nitrite; the acidic environment of the stomach encourages further reduction to create NO and S-nitrosothiols. In conflict with the conclusions of this review, these by-products of nitrites can elicit beneficial health effects; acidified nitrite can enhance the stomach’s antimicrobial activity. Specifically, acidified nitrite has been shown to prevent the growth of Salmonella enteriridis, Salmonella typhumurium, Yersinia enterocolitica, Shigella sonnei, and Escherichia coli (DYK) [91]. NO has also been shown to be a potent vasodilator, and S-nitrosothiols are known to modulate platelet function. These two functions coalesce to improve cardiovascular function, through a lowering of blood pressure. Oxides of nitrogen react with unsaturated fatty acids, such as conjugated linoleic acid, to form nitroalkenes, and these have been shown to inhibit proinflammatory cytokines, such as endothelial TNF-α [92]. The association between processed meat and CRC is more pronounced in those studies considering only nitrite-containing processed meats. However, the relationship still remains modest in comparison to established carcinogens, such as smoking, that exhibit relative risks in excess of 100 fold [93]. Many of the human studies supporting a role for processed meat in colorectal cancer pathogenesis suffer from methodological limitations. Conversely, the preclinical studies are well controlled, yet yield conflicting results. Preclinical studies indicate that that low processed-meat-consumption does not increase CRC risk; however, high consumption does. It is reported that NOC levels are higher in the faeces of rodents consuming nitrite than in controls [17]; however, this increase does not result in an increased incidence Nutrients 2019, 11, 2673 12 of 17 of adenomas, and that is because not all NOCs are carcinogenic [94]. An unintended consequence of the addition of sodium nitrite to meat is the interaction with secondary amines and the formation of NOCs; a wide range of NOCs exist and the individual risk of each is yet to be characterised. It is possible that the discordance in results is explained by a diverse range of NOCs being formed in each preclinical study. The use of nitrites as curing agents is controversial. The findings summarised in this review raise more concerns with its usage. It has been reported that the removal of nitrite from processed meat does not compromise its safety [95]; this issue has attracted considerable media attention [96]. It is clear that well-designed, peer-reviewed research that investigates the efficacy of sodium nitrite as a preservative is needed. Future epidemiological studies should consider more specifically categorising the processed meat consumed, explicitly distinguishing between those meats which are nitrite-containing and those which are not. When this type of data becomes available it will be possible to infer the actual risk posed by the addition of nitrites. Furthermore, preclinical dose response studies are currently lacking and these could help refine the no observed adverse effect level (NOAEL), informing the design of human intervention studies. On the basis of several observational studies [32,34,57,74], the WHO has declared that processed meat should be avoided entirely. Currently, all processed meats are considered group 1 carcinogens, despite there being a vast difference in nutrient compositions. Some processed meats do not contain nitrite; therefore, it is possible that not all processed meats carry the same level of risk.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/11/2673/s1: Table S1: Characteristics of prospective human studies assessing colorectal cancer and processed meat consumption; Table S2: Characteristics of case control human studies assessing colorectal cancer and processed meat consumption. Author Contributions: W.C. conducted the literature searches and composed the tables. W.C. also contributed to the manuscript along with C.T.E. and B.D.G. Funding: Studies investigating links between meat consumption and CRC are in part supported by Agri-Food QUEST, a membership-based, industry-led Innovation Centre for agri-food business in Northern Ireland. Agri-Food QUEST is supported by funding from Invest Northern Ireland. Conﬂicts of Interest: A currently-held Agri-Food Quest project is partially supported by commercial funding from: Finnebrogue Artisan, Karro Food group, and Cranswick. Industrial partners were not involved design or writing of the manuscript, the analysis/interpretation of the data, or the decision to publish.

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