Dietary Nitrosamines from Processed Meat Intake as Drivers of the Fecal Excretion of Nitrosocompounds

Die a y Ni osamines om P ocessed Mea In ake as D i e s o he Fecal Exc e ion o Ni osocompounds Se gio Ruiz-Saa ed a, Tuulia K ee a Pie ilä, Aida Zapico, Cla a G. de los Reyes-Ga ilán, Anne-Ma ia Paja i, and Sonia González* Ci e This: J. Ag ic. Food Chem. 2024, 72, 17588−17598 Read Online ACCESS Me ics & Mo e A icle Recommenda ions ABSTRACT: Die is one o he main exogenous sou ces o po en ially ca cinogenic ni osamines (NAs) along wi h obacco and cosme ics. Se e al ac o s can a ec endogenous N-ni oso compounds (NOCs) o ma ion and he e o e he po en ial damage o he in es inal mucosa a ini ial colo ec al cance s ages. To add ess his issue, 49 olun ee s we e ec ui ed and classi ied acco ding o his opa hological analyses. Li es yle and die a y in o ma ion we e egis e ed a e colonoscopy. The mu agenici y o ecal supe na an s was assayed by a modi ied Ames es . Fecal heme-de i ed NOCs and o al NOC concen a ions we e de e mined by selec i e deni osa ion and chemiluminescence-based de ec ion. Resul s e ealed p ocessed mea s as he main sou ce o die a y ni i es and NAs, iden i ying some o hem as p edic o s o he ecal concen a ion o heme-de i ed and o al NOCs. Fu he mo e, inc eased ecal NOC concen a ions we e ound as he se e i y o colonic mucosal damage inc eased om he con ol o he adenoca cinoma g oup, hese concen a ions being s ongly co ela ed wi h he in ake o he NAs N-ni osodime hylamine, N- ni osopipe idine, and N-ni osopy olidine. Highe ecal NOC concen a ions we e also no ed in highe ecal mu agenici y samples. These esul s could con ibu e o a be e unde s anding o he impo ance o modula ing die a y de i ed xenobio ics as ela ed wi h hei impac on he in es inal en i onmen and colonic mucosa damage. KEYWORDS: ood p ocessing, ni osamines, N-ni oso compounds, in es inal mucosa lesions, hype plas ic polyps, con en ional adenomas, ecal mu agenici y ■INTRODUCTION The consump ion o ed and p ocessed mea has been assessed by he Wo ld Cance Resea ch Fund (WCRF) as a isk ac o o colo ec al cance (CRC) in humans. 1 The p esence o heme i on, he he mal o ma ion o di e en ca cinogens du ing cooking, he gene a ion o lipid and p o ein oxida ion p oduc s, and he o ma ion o exogenous N-ni oso compounds (NOCs) in cu ed mea s o endogenous NOCs in he gas oin es inal ac a e conside ed among he possible mechanisms unde lying his associa ion. 1 Exogenous NOCs mainly comp ised ni osamides and ni osamines (NAs). Ni osamides a e chemically uns able and e en ually decompose. 2 The e o e, he mos abundan NOCs ound in oods a e NAs such as N-ni osodime hylamine (NDMA), N-ni osopipe idine (NPIP), and N-ni osopy oli- dine (NPYR). 3 In his ega d, he Eu opean Food Sa e y Au ho i y has ecen ly issued a echnical epo on he isks o human heal h associa ed wi h he in ake o NAs in oods, concluding ha exposu e o a panel o 10 ca cinogenic NAs aises a heal h conce n. 4 Al hough unp ocessed and uncooked mea may con ain aces o NAs, hese compounds ha e been de ec ed a highe concen a ions in cu ed mea s, smoked ish, cheese, p ese ed ege ables, bee , and human milk, highligh ing he impo ance o cooking and p ocessing me hods in he inal concen a ions. 2 O he nondie a y majo exposu e sou ces o NAs include obacco and pe sonal ca e p oduc s. 4 P e ious s udies in animal and cell models ha e p oposed ha some NAs may be linked o a highe isk o CRC h ough he inc ease o ecal geno oxici y and he o ma ion o highly eac i e diazonium ions, which can gene a e DNA adduc s. 5−7 Fu he mo e, he excess o p o ein e men a ion in he in es ine is associa ed wi h highe le els o amines and o he compounds po en ially oxic o he gu mucosa such as heme i on, which induces he gene a ion o ee adicals in he colon. 8,9 Mos o he a ailable esea ch in humans comes om epidemiological s udies in which he assessmen o NA exposu e is limi ed. The ood equency ques ionnai e (FFQ), a commonly employed me hod o in ake assessmen , es ima es long- e m die a y in ake and enables he compa ison among indi iduals in a popula ion. 10 Howe e , i lacks p ecision and he abili y o e lec he exposu e o o he en i onmen al sou ces o hese compounds such as obacco, wa e , o cosme ic p oduc s. 11 Mo eo e , p edic ed endogenous concen a ions o NOCs ha e been calcula ed o be 100 imes highe han he es ima ed die a y in akes. 12 In he gas oin es inal ac , die a y compo- nen s such as ed mea , p o ein, and NOCs p ecu so s such as ni a es, ni i es, and heme i on can con ibu e o u he ni osa ion p ocesses and he e o e o endogenous o ma ion o Recei ed: June 28, 2024 Re ised: July 23, 2024 Accep ed: July 24, 2024 Published: July 29, 2024 A iclepubs.acs.o g/JAFC © 2024 The Au ho s. Published by Ame ican Chemical Socie y 17588 h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 This a icle is licensed unde CC-BY 4.0 Downloaded ia CSIC on No embe 14, 2024 a 13:15:34 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. ni osyl heme (heme NOCs), S-ni oso hiols (SNOs) and NAs by acid-ca alyzed, in es inal cell o mic obio a media ed pa hways, inally being exc e ed in eces as o al (appa en ) NOCs. 12−16 Howe e , he e a e no s udies ha ha e analyzed he co ela ion be ween he consump ion o hese die a y sou ces and he ecal NOC concen a ions ob ained by analy ical de e mina ions in he con ex o CRC. Based on his e idence, a mo e comp ehensi e app oach is needed o lay he g oundwo k o a be e unde s anding o he complex associa ions be ween die and cance in he long e m. In he p esen s udy, we he e o e in es iga ed he impac o die and die a y xenobio ics on ecal NOC concen a ions and hei associa ion wi h ecal mu agenici y in a sample popula ion o adul s a di e en s ages o in es inal mucosa damage in he p og ession o CRC, a e biopsy examina ion. ■MATERIALS AND METHODS S udy Design and Volun ee s. This s udy is pa o b oade p ojec s ela ed o he e ec o die and die a y xenobio ics on in es inal mucosa and ela ed gu mic obio a p o iles in he con ex o CRC (MIXED and MiToxicDie p ojec s). The ec ui men o olun ee s was ca ied ou om Oc obe 2019 o Decembe 2021 by Facul a i es o he Diges i e Se ice om he Cen al Uni e si y Hospi al o As u ias (HUCA) and he Ca men and Se e o Ochoa Hospi al in Cangas de Na cea, As u ias, Spain. Volun ee s we e selec ed among indi iduals en olled in a colon cance sc eening p og am. Inclusion c i e ia we e being be ween 40 and 79 yea s old and no e e ing o he in ake o omep azole, an ibio ics, co icoids, o nons e oidal an i- in lamma o y d ugs. In addi ion, ha ing speci ic cance ea men a he ime o he s udy o in he p e ious 2 mon hs, p e ious su ge y o he diges i e sys em, au oimmuni y, al e ed hy oid unc ion, o his o y o diabe es o goi e we e exclusion c i e ia. Those indi iduals in e es ed in pa icipa ing we e in o med o he objec i es o he s udy and signed an in o med consen o m. A o al o 49 subjec s we e included in he s udy. Pa ien s we e asked o p o ide a s ool sample collec ed p io o p epa a ion o colonoscopy. Du ing he p ocedu e, a biopsy o he emo al o issue samples was pe o med. Biopsies we e examined a he Depa men o Ana omical Pa hology o HUCA, as desc ibed elsewhe e. 17 A e biopsy examina ion pa ien s we e classi ied in o ou his opa hology g oups in o de o inc easing isk o CRC de elopmen : nonpa hological con ol (NP) (n= 18), hype plas ic polyps (HP) (n= 10), con en ional adenomas (CA) (n= 18), and adenoca cinomas (AC) (n= 3). This p ojec was e alua ed and app o ed by he Regional E hics Commi ee o Clinical Resea ch o As u ias ( e . 163/19) and by he Commi ee on Bioe hics o CSIC ( e . 174/2020). The p ocedu es we e pe o med in acco dance wi h he undamen al p inciples se ou in he Decla a ion o Helsinki, he O iedo Bioe hics Con en ion, and he Council o Eu ope Con en ion on Human Righ s and Biomedicine, as well as in Spanish legisla ion on bioe hics. Di ec i e 95/46/EC o he Eu opean Pa liamen and he Council o Oc obe 1995, on he p o ec ion o indi iduals ega ding he p ocessing o pe sonal da a, was s ic ly ollowed. Nu i ional Assessmen . Die a y in o ma ion was ob ained om olun ee s h ough a pe sonalized in e iew conduc ed by a ained in e iewe when hey we e in o med abou he colonoscopy esul s a medical consul a ion. Excep ionally, as a esul o he COVID-19 es ic ion o isi o s o hospi als in Spain du ing he pandemic, some o he su eys we e conduc ed h ough online ools. Fo his pu pose, a 155-i em semiquan i a i e FFQ p e iously de eloped by he esea ch g oup and alida ed o he es ima ion o die a y xenobio ics in ake was used. 18 In addi ion o ood and culina y p epa a ions, he speci ic ype o ood was eco ded, as well as cooking and p ocessing me hods and o he ela ed ques ions such as he deg ee o doneness o empe a u e, when necessa y. In o ma ion ela i e o die a y assessmen o he es ima ion o he die a y in ake o xenobio ics has been p e iously published. 17 The classi ica ion o oods in o ood g oups was ca ied ou acco ding o he Cen e o Highe Educa ion in Nu i ion and Die e ics (CESNID) c i e ia. 19 Then, ood composi ion ables o CESNID we e used o ans o m ood consump ion in o ene gy, mac o- and mic onu ien in ake. 19 The con en o ni a es, ni i es, and he exogenous NAs NDMA, NPIP, and NPYR was es ima ed using he Eu opean P ospec i e In es iga ion in o Cance and Nu i ion (EPIC) Po en ial Ca cinogen Da abase and o he da abases p e iously indica ed. 17,20 Speci ically, NA concen a ion alues we e compiled o p ocessed mea s ypically consumed in he geog aphical egion, such as cu ed ham (po k mea ), cho izo (a Spanish sausage o minced po k mea combined wi h pap ika and spices), and blood sausage (made o cooked blood gene ally ob ained om po k, po k a , onion, and spices). 21,22 Ou o he 74 oods in he inal da abase consis ing o 258 di e en i ems ega ding ni a es, ni i es, and NAs concen a ion alues, 54 oods we e consumed in he sample. O hese, 27 we e ege able p oduc s, and 10 we e p ocessed mea s. The phenolic con en o he oods was ex ac ed om Phenol Explo e 3.6 and ibe con en om he ables by Ma le and Cheung. 23,24 Oxygen Radical Ac i i y Capaci y (ORAC) was calcula ed based on he a icle by Wu e al. 25 Fo each die a y compound o in e es , he ood sou ces wi h a leas 5% con ibu ion in each case we e conside ed. Heme i on in ake was calcula ed by assuming ha heme i on was 40% o o al i on con ained in all mea s, ish, and eggs, as p oposed by Monsen and Balin y. 26 Die a y in o ma ion was ob ained o 36 olun ee s om NP (n= 11), HP (n= 8), CA (n= 14), and AC (n= 3) g oups. Gene al Cha ac e is ics. Du ing pe sonalized in e iews, sleeping hou s and physical ac i i y we e eco ded as he a e age sel - e e ed ime pe day o each indi idual conside ing he las yea , while he numbe o deposi ions was eco ded as he sel - e e ed imes du ing a no mal week. In o ma ion on smoking habi s was ob ained by asking abou ciga e e smoking h oughou li e. The an h opome ical pa ame e s heigh (m) and weigh (kg) we e aken by s anda dized p o ocols. 27 Body mass index (BMI) was calcula ed using he o mula weigh /(heigh )2. Measu emen o Fecal To al NOCs and Heme NOCs. Fecal homogena es we e p epa ed by dilu ing samples (1:5) wi h ul apu e Milli-Q wa e ( esis i i y 18,2 MΩcm a 25 °C, Millipo e, Rios30) and homogenized wi h T-18 Digi al Ul a Tu ax (IKA, Ge many). To al NOCs and heme NOCs we e analyzed om he ecal homogena es using selec i e deni osa ion and chemiluminescence-based de ec ion by Ecomedics CLD 88 Exhalyze (Eco Medics, Swi ze land) equipped wi h a cus om-made liquid pu ge essel and a NaOH (1 mol/L, kep a 4°C) ap as p esen ed in p e ious s udies. 28,29 Unless o he wise s a ed, he chemicals we e ob ained om Sigma-Ald ich (Me ck Li e Science). P ese a ion solu ion [250 mg o N-e hylmaleimide (M= 125 g/ mol), and 78 mg o die hylene iaminepen aace ic acid (M= 393 g/ mol) in 20 mL o Milli-Q wa e ] was used o p e en a i icial ni osa ion ia binding o me al i on and alkyla ing ee hiol g oups, whe eas acid sulphanilamide (SA) solu ion [5 g/100 mL in 1 M hyd ogen chlo ide (HCl)] was used o emo e ni i e. 30 Use o selec i e deni osa ion enables he indi ec de e mina ion o heme NOCs. The e o e, me cu y(II) and e icyanide s able ni oso compounds (nonheme) we e de e mined using aqueous HgCl2(10 mM HgCl2,M = 271.5 g/mol) and K3Fe(CN)6(10 mM K3Fe(CN)6,M= 368.35 g/ mol). Fecal homogena e (100 μL) was incuba ed a oom empe a u e o 5 min wi h 100 μL p ese a ion and 500 μL o SA solu ion wi h o wi hou HgCl2and K3Fe(CN)6(100 μL/each). Fi y mic oli e s we e injec ed in o he pu ge essel con aining 15 mL o educing agen iiodine mix u e [1 g po assium iodine (M= 166.0 g/mol) and 0.65 g iodine (M= 253.81 g/mol, Ac os O ganics, UK)], 70 mL glacial ace ic acid (WWR Chemicals, F ance), 20 mL Milli-Q wa e , and An i oam B Emulsion (#A6707) kep a 60 °C ha eleases ni ogen oxide (NO) om NAs, SNOs, alkyl ni i es, and i on ni osyl compounds. Sys em helium gas low (110 mL/min, adjus ed 0 ±5.0 mba ) mixes he sample and ans e s he eleased NO o he CLD 88 ia a condense , he chemical ap, and a 0.20 μm polyp opylene il e . Each sample was injec ed wice, i s o o al NOC and hen o heme NOC de e mina ion. 5−10 min we e le in be ween he injec ions o he signal o e u n o baseline. The iiodine mix u e was changed a in e als o h ee samples. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17589 The signal was eco ded wi h a Powe Ch om 280 sys em and analyzed by he ins umen so wa e (Powe Ch om, eDAQ, Aus alia). Cu o equency o he signal was se o 0.05 Hz o educe he noise. Calib a ion cu es o known s anda ds (5 o 950 picomoles, pmol) o Table 1. Gene al Desc ip ion o he S udy Sample Acco ding o His opa hological Classi ica ion a WS (n= 36) NP (n= 11) HP (n= 8) CA (n= 14) AC (n= 3) gende male 15 (41.7%) 3 (27.3%) 3 (37.5%) 6 (42.9%) 3 (100.0%) emale 21 (58.3%) 8 (72.7%) 5 (62.5%) 8 (57.1%) 0 (0.0%) age (yea s) 61 ±7 59 ±9 59 ±7 62 ±6 64 ±3 weigh (kg) 73.51 ±14.49 71.14 ±15.93 74.63 ±13.62 74.57 ±16.23 74.33 ±1.15 heigh (cm) 166.74 ±9.79 165.18 ±12.37 169.25 ±7.23 164.75 ±9.24 175.00 ±2.65 BMI (kg/m2) 26.25 ±3.95 25.66 ±4.04 25.97 ±4.21 27.30 ±4.18 24.28 ±0.37 ene gy in ake (kcal/day) 1993.30 ±812.16 1830.12 ±779.93 1979.17 ±1005.41 2103.66 ±837.09 2114.34 ±393.01 e hanol (g/day) 11.40 ±20.38 2.93 ±5.69 17.49 ±30.16 16.23 ±21.79 *3.68 ±6.38 smoking habi cu en 4 (11.1%) 0 (0.0%) 2 (25.0%) 1 (7.1%) 1 (33.3%) ne e 20 (55.6%) 7 (63.6%) 5 (62.5%) 7 (50.0%) 1 (33.3%) o me 12 (33.3%) 4 (31.3%) 1 (12.5%) 6 (42.9%) 1 (33.3%) sleeping (h/day) 7.11 ±1.05 7.10 ±0.74 7.25 ±1.28 7.07 ±1.21 7.00 ±1.00 physical ac i i y (min/day) 57.29 ±25.62 59.32 ±21.33 64.69 ±23.35 45.54 ±27.35 85.00 ±8.66 deposi ions ( imes/week) 6.85 ±2.26 6.91 ±2.19 6.13 ±2.74 7.04 ±2.29 7.67 ±1.44 a Only indi iduals wi h in o ma ion on ecal NOC concen a ion and die a e included in he able. Values a e p esen ed as mean ±SD o con inuous a iables o numbe (%) o ca ego ical ones. (*) S a is ically signi ican di e ences compa ed o he NP g oup (p< 0.05) we e ound wi h he Mann−Whi ney U es . NOCs, N-ni oso compounds; BMI, body mass index; WS, whole sample; NP, nonpa hological con ols; HP, hype plas ic polyps; CA, con en ional adenomas; AC, adenoca cinomas. Figu e 1. Majo die a y sou ces o ni a es, ni i es, and NAs in (A) he sample popula ion o (B) acco ding o emale o male gende . NAs, ni osamines; NDMA, N-ni osodime hylamine; NPIP, N-ni osopipe idine; NPYR, N-ni osopy olidine; W/s, wi hou speci ying. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17590 aqueous sodium ni i e (NaNO2) we e used o quan i ica ion by compa ing he a ea unde he cu e o he a ea o known s anda ds. Calib a ion cu e de e mina ion coe icien (R2) > 0.96 was accep ed. Aliquo s o a pooled sample we e used as he in e nal con ol o moni o ing in e day ep oduc i i y o he sys em and calib a ion cu es p io o injec ing samples. Peak selec ion was done based on isual e alua ion and he de e mina ion o he quan i ica ion limi was se o 2.5 pmol. Heme NOCs we e de e mined by sub ac ing he alues o me cu y(II) and e icyanide s able compounds om he o al NOC. Concen a ions we e ob ained o 49 olun ee s om NP (n= 18), HP (n= 10), CA (n= 18), and AC (n= 3) g oups and exp essed as pmol pe millig am o ecal sample (pmol/mg). Fecal Mu agenici y. The Ames es assayed he mu agenici y o ecal supe na an s wi hou me abolic ac i a ion agains he s ain Salmonella en e ica se o a Typhimu ium TA100. The 5051 Mu a- Ch omoPla eTM ki (EBPI, On a io, USA) was used. Fil e ed ecal supe na an dilu ions we e mixed wi h he bac e ia g own o e 16 h a 37 °C in he s e ile medium p o ided by he manu ac u e and he solu ion mix con aining Da is-Mingoli sal s, D-glucose, b omoc esol pu ple, D-bio ine, and L-his idine as indica ed by he manu ac u e . Posi i e con ol (including sodium azide as a mu agen, g own bac e ia, and solu ion mix), nega i e con ol (including only solu ion mix), and he app op ia e se ies o dilu ions o ecal supe na an s we e added o 96-well mic o i e pla es con aining 200 μL pe well and incuba ed a 37 °C o 5 days. Re e sion a es (RR) we e calcula ed o condi ions whe e less han 96 e e an wells pe pla e and mo e han 48 e e an wells in he posi i e con ol we e ob ained. Conside ing he dilu ion ac o , he le els o mu agenici y we e exp essed as he mean o alues co esponding o he h ee dilu ions es ed pe sample and we e a bi a ily classi ied as “low” [180−299] (n= 6), “medium” [300−599] (n= 27) o “high” [600−1474] (n= 8) mu agenici y. 17 The in e e ence wi h L-his idine du ing he mu agenici y assay was uled ou as indica ed in Ruiz-Saa ed a e al. 17 Fecal mu agenici y alues we e ob ained o 41 olun ee s om NP (n= 15), HP (n= 9), CA (n= 14), and AC (n= 3) g oups. S a is ical Analyses. Resul s we e analyzed using he IBM SPSS so wa e e sion 25.0 (IBM SPSS, Inc., Chicago, IL, SA). G aphPad P ism 9, RS udio e sion 1.4.3, and BioRende so wa e we e used o g aphical ep esen a ions. O e all, ca ego ical a iables we e summa - ized as numbe s and pe cen ages and con inuous ones as mean and s anda d de ia ion. Fishe es s we e pe o med o ca ego ical a iables (p- alue <0.05). Fo con inuous a iables, he goodness o i o a no mal dis ibu ion was checked by means o he Kolmogo o − Smi no es . When no mali y o a iables was achie ed, T- es s we e pe o med; o he wise, Mann−Whi ney U es s we e applied (p- alue <0.05). Spea man co ela ions we e ca ied ou o explo e he associa ions be ween he in ake o ood g oups, oods, die a y compounds, and he ecal NOC concen a ion. Hea maps we e gene a ed using “co plo ” R package. Acco ding o WCRF, a cu o in ake alue o 50 g o p ocessed mea pe day was selec ed o analyze di e ences in ecal NOC concen a ions. 1 The ela ionship be ween ecal NOC concen a ions and ecal mu agenici y was e alua ed by pe o ming simple linea eg essions. ■RESULTS Gene al Cha ac e is ics o he Sample Popula ion. The gene al cha ac e is ics o he human s udy sample acco ding o pos colonoscopy his opa hological diagnosis and nu i ional assessmen a e p esen ed in Table 1. The gene al sample is mos ly composed o women (58%), he age a e age was 61 yea s old, wi h a mean BMI o 26.25 kg/m2, which indica es o e weigh ; mos o he olun ee s we e nonsmoke s (56%). Ou o he o al sample, 31% we e NP in compa ison wi h 22% in he HP g oup, 39% displaying CA, and 8% diagnosed wi h AC. The in ake o e hanol was signi ican ly highe in he CA g oup in compa ison wi h he NP g oup (16.23 g/day s 2.93 g/ day). No s a is ically signi ican di e ences we e ound o he es o he a iables analyzed be ween NP and HP, CA, o AC g oups. Die a y Sou ces o Ni a es, Ni i es, and Exogenous NOCs (NAs) and Thei Associa ion wi h Fecal NOCs. The majo oods con ibu ing o he in ake o ni a es, ni i es, and he NAs NDMA, NPIP, and NPYR in he o al sample a e shown in Figu e 1A. While he in ake o ni a es in he sample is explained by he consump ion o oo s and ege ables (mainly 27% le uce, 11% po a o, 11% cha d, 8% whi e onion, 8% spinach, 6% cabbage, and 5% squash), ni i es and NAs mainly de i e om he consump ion o p ocessed mea s (such as bacon, ham, o cho izo). Eggs and some plan oods such as po a oes, spinach, onion, and cucumbe also con ibu ed as a whole o 14% o he die a y in ake o ni i e, al hough hey we e ep esen ed all oge he in he ca ego y “o he oods” in Figu e 1A as hei indi idual con ibu ion was lowe han 5%. Fu he mo e, 12% o he in ake o NDMA is p o ided by bee . Di e ences in he die a y sou ces o he compounds unde s udy we e also analyzed by gende , and esul s a e p esen ed in Figu e 1B. Sou ces o ni a e we e simila o bo h gende s wi h he excep ion o squash and cabbage, only iden i ied as a ni a e die a y sou ce in emales. Rega ding he die a y sou ces o ni i es, in bo h gende s, he main con ibu o s we e cu ed and cooked ham. Bacon also con ibu ed (>5%) o he in ake o his compound, bu only in men. In u n, bo h cu ed and cooked ham oge he wi h cho izo accoun ed o mos o he in ake o NDMA, NPIP, and NPYR in bo h gende s as well as blood sausage exclusi ely in males. No di e ences in he in ake o ni a es, ni i es, and he di e en NAs acco ding o his opa hology g oups we e de ec ed (Table 2). A Spea man co ela ion analysis was conduc ed o u he explo e he associa ion be ween he in ake o he majo die a y sou ces o ni a es, ni i es, and NAs and he ecal concen a ion o NOCs (Table 3). F om he assessed oods, cha d and spinach we e nega i ely co ela ed wi h he heme NOCs, con a y o o he oods, including po a o, bacon, cu ed ham, cho izo, and blood sausage ha we e posi i ely co ela ed wi h he ecal o al NOCs and wi h heme NOCs. A s epwise eg ession analysis was Table 2. Die a y In akes o Ni a es, Ni i es, and NAs Acco ding o His opa hology G oups a WS (n= 36) NP (n= 11) HP (n= 8) CA (n= 14) AC (n= 3) ni a es (mg/day) 127.02 ±117.03 171.86 ±166.81 88.3 ±47.57 112.04 ±101.29 135.8 ±84.88 ni i es (mg/day) 3.13 ±2.32 2.91 ±1.75 3.02 ±3.05 3.57 ±2.58 2.23 ±0.44 NDMA (μg/day) 0.20 ±0.17 0.14 ±0.1 0.23 ±0.25 0.23 ±0.17 0.17 ±0.06 NPIP (μg/day) 0.09 ±0.07 0.08 ±0.05 0.09 ±0.09 0.10 ±0.07 0.06 ±0.02 NPYR (μg/day) 0.14 ±0.10 0.13 ±0.09 0.13 ±0.14 0.16 ±0.11 0.10 ±0.03 a Values a e p esen ed as mean ±SD. No s a is ically signi ican di e ences we e ound (p< 0.05) wi h he Mann−Whi ney U es . NAs, ni osamines; WS, whole sample; NP, nonpa hological con ols; HP, hype plas ic polyps; CA, con en ional adenomas; AC, adenoca cinomas; NDMA, N-ni osodime hylamine; NPIP, N-ni osopipe idine; NPYR, N-ni osopy olidine. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17591 pe o med o de e mine which o hese ood sou ces co ela ing wi h ecal o al and heme NOC concen a ions we e p edic o s o hese a iables. “Cho izo, ca ego y w/s” was e ealed as one o he ood sou ces p edic ing he le els o ecal o al and heme NOCs in his sample (Table 4). Fecal NOCs (To al and Heme NOCs) Concen a ions Acco ding o His opa hology G oups. The di e ences in ecal NOC concen a ions acco ding o he his opa hology g oups a e shown in Figu e 2. Ou da a showed an inc ease in he ecal concen a ion o o al NOCs and heme NOCs as he g ade o in es inal mucosa lesion inc eases om NP o AC, ob aining s a is ically signi ican di e ences be ween hese wo ex eme g oups (NP and AC) o o al NOCs (6.50 s 20.69 pmol/mg o eces, p= 0.006) and heme NOCs (4.40 s 16.80, p = 0.017). Al hough his end was main ained in each Table 3. Spea man Co ela ions be ween he In ake o he Majo Food Sou ces o Ni a es, Ni i es, and NAs and he Concen a ion o Fecal NOCs in he Sample Popula ion a ood in ake (g/day) ni a e (mg/100 g ood) ni i e (mg/100 g ood) NDMA (μg/100 g ood) NPIP (μg/100 g ood) NPYR (μg/100 g ood) compound Rho (Spea man) p alue po a o 52.89 ±30.41 16.8 0.11 0 0 0 To al NOCs 0.431 0.009 Heme NOCs 0.437 0.008 cha d 14.99 ±42.45 203.0 0.13 0 0 0 To al NOCs −0.274 0.106 Heme NOCs −0.354 0.034 spinach 9.46 ±17.62 163.0 0.77 0 0 0 To al NOCs −0.247 0.146 Heme NOCs −0.358 0.032 po k, bacon 2.95 ±6.52 3.2 10.1 1.01 0 0 To al NOCs 0.524 0.001 Heme NOCs 0.517 0.001 cu ed ham, ull a 26.19 ±25.43 2.9 7.20 0.20 0.18 0.29 To al NOCs 0.364 0.029 Heme NOCs 0.392 0.018 cho izo, ca ego y w/s 11.99 ±17.18 0 0 0.20 0.08 0.12 To al NOCs 0.468 0.004 Heme NOCs 0.491 0.002 blood sausage 3.91 ±9.42 0 0 0.35 0.20 0.21 To al NOCs 0.433 0.008 Heme NOCs 0.419 0.011 a In ake alues a e p esen ed as mean ±SD. Ni a e, ni i e, NDMA, NPIP, and NPYR concen a ion alues in oods we e ob ained om EPIC and o he au ho s' da a. 17,20−22 Only die a y sou ces showing s a is ically signi ican Spea man co ela ion p- alue <0.05 in a leas one ecal NOC a iable a e shown. NAs, ni osamines; NOCs, N-ni oso compounds; W/s, wi hou speci ying. Table 4. Resul s Ob ained om S epwise Reg ession Analyses Iden i ying Food Sou ces o Ni a es, Ni i es, and NAs P edic o s o Fecal NOCs Concen a ion a dependen a iable independen a iables R2βp alue included* o al NOCs po a o 0.317 0.052 No po k, bacon −0.201 0.205 No cu ed ham, ull a −0.250 0.165 No cho izo, ca ego y w/s 0.152 0.420 0.011 Yes blood sausage 0.192 0.254 No heme NOCs po a o 0.273 0.095 No cha d −0.100 0.539 No spinach −0.205 0.205 No po k, bacon −0.227 0.149 No cu ed ham, ull a 0.271 0.130 No cho izo, ca ego y w/s 0.156 0.424 0.010 Yes blood sausage 0.167 0.319 No a Only he a iables wi h p< 0.05 in p e ious co ela ion analyses a e included in he model. *Independen a iables showing p< 0.05 a e inally included in he s epwise eg ession. NAs, ni osamines; NOCs, N-ni oso compounds; R2, coe icien o mul iple de e mina ion; β, s anda dized eg ession coe icien . Figu e 2. Di e ences in ecal NOCs concen a ion acco ding o his opa hology g oups. Ba s ep esen he mean concen a ion and e ical lines he s anda d de ia ion wi hin each his opa hology g oup. (*) S a is ically signi ican di e ences we e ob ained be ween his opa hology g oups (Mann−Whi ney U es , p< 0.05). NOCs, N- ni oso compounds; NP, nonpa hological con ols; HP, hype plas ic polyps; CA, con en ional adenomas; AC, adenoca cinoma. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17592 his opa hology g oup, he di e ences we e no s a is ically signi ican . S a is ically signi ican highe ecal NOC concen a ions we e also obse ed in olun ee s consuming >50 g/day o p ocessed mea , a ood g oup ha included di e en oods, such as bacon, sausages, and cu ed ham and e men ed mea s, such as cho izo and blood sausage, in compa ison wi h olun ee s consuming <50 g/day, wi h o al NOC concen a ions o 12.78 and 7.39 pmol/mg o eces, espec i ely (p= 0.044), and heme NOC concen a ions o 9.62 and 4.58, espec i ely (p= 0.017). Focusing on p ocessed mea , a close examina ion o associa ions be ween ecal NOC concen a ions and die a y in ake e ealed in e g oup di e ences (Figu e 3). The whole sample p esen s posi i e associa ions be ween he in ake o p ocessed mea s, ed mea , ce eal p oduc s, and po a oes wi h heme NOC concen a ions. The same co ela ions o he whole sample appea ed a highe in ensi y in he NP as compa ed wi h he HP g oup. Speci ically, he NP g oup displayed a signi ican posi i e associa ion o ish consump ion wi h o al NOCs and heme NOCs, whe eas he same was ue o ege ables in he HP g oup (Figu e 3A). In con as , nega i e co ela ions o sea ood and snacks wi h ecal NOCs we e obse ed in he CA g oup; his g oup main ained he same posi i e associa ion ound in he o he g oups o he in ake o p ocessed mea and ecal NOCs al hough hese only eached s a is ical signi icance o he in ake o po k bacon. In Figu e 3B, he associa ions ound be ween die a y compounds and ecal NOCs a e depic ed. Al hough he whole sample p esen ed nega i e co ela ions o he in ake o phenolic acids and o al polyphenols wi h ecal heme NOC concen a ion, he die a y NDMA, NPIP, o NPYR we e posi i ely co ela ed wi h o al and heme ecal NOCs. Rega ding he di e en his opa hological g oups, in e se associa ions we e ound o he NP g oup be ween he in ake o ni a es and la onoids wi h ecal heme NOC concen a ions, whe eas posi i e associa ions we e ound be ween he in ake o o al p o ein and mic onu ien s, such as i amin B12, i amin D, phospho us, and selenium wi h ecal o al and heme NOCs. No signi ican associa ions we e ound in he case o he CA g oup. Heme i on de i ed om mea , ish, and eggs in ake was posi i ely co ela ed wi h o al and heme ecal NOC Figu e 3. Hea maps de ined by Spea man co ela ions acco ding o his opa hology g oups be ween ecal NOCs and (A) ood g oups and oods o (B) die a y compounds. Blue and ed colo s ep esen nega i e and posi i e associa ions, espec i ely. The colo in ensi y is p opo ional o he deg ee o associa ion. (*)p< 0.05 (**)p< 0.01. NOCs, N-ni oso compounds; WS, whole sample; NP, nonpa hological con ols; HP, hype plas ic polyps; CA, con en ional adenomas; AC g oup was no included in he analysis as a esul o he limi ed sample size (n= 3); I, insoluble; S, soluble; ORAC, oxygen adical abso bance capaci y; NDMA, N-ni osodime hylamine; NPIP, N-ni osopipe idine; NPYR, N-ni osopy olidine. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17593 concen a ions in he whole sample ( = 0.364 and = 0.374, espec i ely) and NP g oup ( = 0.645 and = 0.745, espec i ely) bu no in HP and CA g oups. Rela ionships among Fecal NOCs and Fecal Mu a- genici y. The concen a ion o ecal NOCs was e alua ed acco ding o he ecal mu agenici y g oup (Table 5). No s a is ically signi ican di e ences we e ob ained in he concen a ion o hese compounds as a unc ion o mu agenici y le els, al hough highe o al and heme NOCs we e obse ed in he high mu agenici y g oup. The ela ionship was u he examined by linea eg ession analysis (Figu e 4). When he whole sample was conside ed, he ecal concen a ions o o al NOCs (R2= 0.117, p< 0.05) and heme NOCs (R2= 0.167, p< 0.05) inc eased as ecal mu agenici y inc eased. The NP g oup p esen ed he sligh es posi i e associa ion be ween ecal NOCs and mu agenici y in compa ison wi h he es o he g oups. In he case o he HP g oup, a signi ican associa ion be ween ecal heme NOCs and mu agenici y was ound (R2= 0.453, p< 0.05). ■DISCUSSION To he bes o ou knowledge, his is he i s s udy in es iga ing he con ibu ion o die and i s componen s, wi h special emphasis on NAs de i ed om ood p ocessing and on endogenous NOC o ma ion in humans in he con ex o CRC. In his wo k, g ea e concen a ions o ecal o al NOCs and heme NOCs we e ound as he se e i y o colonic mucosa damage inc eased. Among he p oposed mechanisms o explain he associa ion be ween CRC isk and highe ed and p ocessed mea consump ion is he endogenous o ma ion o possible ca cinogenic NOCs h ough he gas oin es inal ac . 14,15,31,32 The ecal concen a ion o NOCs depends on many ac o s such as he in ake o exogenous NOCs, ni a e, and ni i es ha could ac as ni osa ing agen s, amines, and amides ha could be ans o med in o seconda y amines, heme i on om ood and he hos mic obio a (Figu e 5). 13,33,34 App oxima ely 7% o die a y ni a es can be educed o ni i e by bac e ial ni a e educ ase in he o al ca i y, o which he heme g oup ac s as an elec on dono a o ing he ca aly ic o ma ion o ni i e. 35 Once in he s omach, ni i e is ans o med o ni ous acid due o he low pH condi ions and may lead o he o ma ion o NAs and SNOs a e eac ion wi h seconda y amines and hiol g oups ob ained om p o eolysis o p o ein ood. 13,36 SNOs acili a e NO elease and he ni o- syla ion o heme i on om mea sou ces in he small in es ine. 37 Ni osa ing agen s o med om NO p oduced by inducible NO syn hase (iNOS) in colonocy es oge he wi h ni a e, ni i e, heme NOCs, SNOs, and in es inal mic obio a could con ibu e o he u he o ma ion o endogenous NOCs in his loca ion. 13,38,39 Many NAs a e ca cinogenic compounds, causing damage h ough DNA al e a ion. 40 In addi ion, he heme g oup can inc ease he ac i i y o ni osa ing agen s and can con ibu e o DNA damage by inc easing lipid pe oxida ion and gene a ion o cy o oxic and geno oxic aldehydes. 41−43 Table 5. Fecal NOCs Concen a ions Acco ding o Fecal Mu agenici y G oups a (pmol/mg o eces) low mu agenici y (0−300)(n= 6) medium mu agenici y (>300−600)(n= 27) high mu agenici y (>600) (n= 8) o al NOCs 11.05 ±7.76 9.44 ±9.76 15.85 ±12.68 heme NOCs 7.81 ±6.29 6.27 ±7.78 12.52 ±10.70 a Values a e p esen ed as mean ±SD. No s a is ically signi ican di e ences we e ound (p< 0.05) wi h he Mann−Whi ney es . NOCs, N-ni oso compounds. Figu e 4. Linea eg essions o ecal NOCs depend on he ecal mu agenici y alues and acco ding o his opa hology g oups. NOCs, N-ni oso compounds; WS, whole sample; NP, nonpa hological con ols; HP, hype plas ic polyps; CA, con en ional adenomas; AC, adenoca cinomas. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17594 Simila o wha occu s in mos Wes e nized socie ies, he a e age consump ion o ed (58.27 g/day) and p ocessed mea (64.83 g/day) in he s udy sample was abo e he Spanish ecommenda ions o a maximum o 500 g pe week (app oxima ely 71 g/day o combined ed and p ocessed mea ). 44 In addi ion, he mean in ake o p ocessed mea in he sample o he s udy was abo e he isk dose o CRC (50 g/ day). 1 Mo eo e , olun ee s showing p ocessed mea in akes >50 g/day p esen ed highe ecal concen a ions o o al and heme NOCs. Ou indings poin ed o p ocessed mea s ich in ni i es and NAs, such as bacon, ull a cu ed ham, cho izo (a Spanish cu ed sausage), and blood sausage, as he main die a y sou ces o NOCs associa ed wi h ecal o al NOCs and heme NOCs. In ou esul s, bee appea ed as a die a y sou ce o NDMA based on a e e ence om an EPIC s udy ha da ed back o 1988. 45 P ocedu es and s anda ds in he ood indus y ha e imp o ed no ably in he las decades, which p obably has con ibu ed o educe he o iginally epo ed concen a ion o NDMA in e men ed be e ages. In e es ingly, po a o in ake was ound o be co ela ed wi h ecal NOCs, p obably because hey a e o en consumed wi h mea , sugges ing a con ounding e ec as p e iously epo ed by o he au ho s. 46 Among all ood sou ces s udied in his wo k, cho izo was a p edic o o he le els o o al ecal NOCs in he sample s udied. Al hough he esul was s a is ically signi ican , he low R2 alue ob ained in he model sugges ed ha o he ac o s no conside ed in his wo k, such as he in es inal mic obiome, could be playing an impo an ole in NOC o ma ion. I is also no ewo hy ha he in ake o phenolic acids and o al polyphenols was nega i ely co ela ed wi h ecal heme NOCs, sugges ing an inhibi o y e ec o hese plan -de i ed bioac i es on endogenous NOC o ma ion. Case-con ol s udies a e o en in luenced by selec ion, ecall, and epo ing biases when die a y in ake assessmen is done a e diagnosis. Howe e , since in es inal polyps a e o en asymp oma ic, i is less likely ha pa ien s ha e al e ed hei die a y in ake be o e he isi o he hospi al. The me hodology used o egis a ion o he die a y in ake and he u he da abases employed o con e sion in o di e en die a y compounds wi h ca cinogenic and bioac i e e ec s ha e allowed us o ob ain a high deg ee o de ail on die a y in ake in o ma ion. Howe e , di e ences in he in ake o ni a es, ni i es, NDMA, NPIP, and NPYR among he ew s udies a ailable in he li e a u e a e o be expec ed due o he di e en me hods o die a y analysis used in each case. In his ega d, Hol op e al. de e mined ha die composi ion was associa ed wi h he endogenous o ma ion o NOCs in a sample popula ion o obese men. Fo ha pu pose, hey used he McCance and Widdowsons’ ables wi h semiquan i a i e da a om di e en ood ca ego ies ins ead o using a de ailed es ima ion o he in ake o each indi idual ood i em. 47 The use o die a y his o y ques ionnai es epo ed lowe mean in akes o ni i es (0.99 s 3.13 mg/day in ou s udy) and NDMA (0.114 μg/day s 0.198 μg/day in ou s udy) in a Spanish popula ion as compa ed o he in akes ob ained nea ly 30 yea s ago in a Finnish sample popula ion (5.30 mg/day o ni i e and 0.05 μg/day o NDMA). 48,49 Rega ding he in ake o NPYR and NPIP, p e ious s udies a e sca ce. In ou sample, we de ec ed an in ake o 0.138 and 0.088 μg/day o NPYR and NPIP, espec i ely. Die a y es ima ions om Ge man nu i ional su eys se NPYR and NPIP exposu e o 0.011 and 0.015 μg/day, espec i ely, bu his s udy did no de ail each mea consump ion le el, which could ha e helped o be e unde s and he di e ences be ween he esul s. 50 One o he main objec i es o his wo k was o de e mine whe he he es ima ion o die a y ni a es, ni i es, and NAs could be ep esen a i e and could display a meaning ul ela ionship wi h he concen a ions o NOCs exc e ed. The whole sample p esen ed nonsigni ican co ela ions be ween he Figu e 5. Schema ic ep esen a ion o he mechanisms and die a y ac o s a ec ing endogenous NOCs o ma ion and exc e ion o he s ool. G ay a ows indica e die a y sou ces o p ecu so s and induce s o NOCs o ma ion and black a ows indica e endogenous NOCs o ma ion. NOCs, N- ni oso compounds; NO3 −, ni a e ion; NO2-, ni i e ion; HNO2, ni ous acid; Sec, seconda y; NAs, ni osamines; SNOs, S-ni oso hiols; Heme NOCs, ni osyl heme; NO, ni ogen monoxide; iNOS, inducible NO syn hase. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17595 in ake o ni a es and ni i es, espec i ely, and ecal NOC concen a ions. Howe e , he in ake o NDMA, NPIP, NPYR, and heme i on om mea s, eggs, and ish was signi ican ly and posi i ely co ela ed wi h ecal o al and heme NOCs, in conco dance wi h p e ious s udies ha poin ed o hei di ec associa ion wi h NOC o ma ion. 13 Al hough he s a is ical signi icance o hese associa ions was no main ained wi hin he g oups es ablished acco ding o he damage o he colo ec al mucosa, p obably due o he limi ed sample size and dispe sion o he da a, simila ends we e no ed in he case o he ood die a y sou ces o hese compounds. Speci ically, heme NOCs we e posi i ely and signi ican ly associa ed wi h he in ake o bacon and cu ed ham in he NP g oup and wi h bacon in he CA g oup, which also showed inc eased consump ion o e hanol. Mo eo e , we ound ha i amin C, an an ioxidan molecule p e iously desc ibed as an inhibi o o NOC o ma ion, was posi i ely co ela ed wi h ecal o al and heme NOCs in he HP g oup, p obably because o he s ong co ela ion o ege able consump ion wi h ecal NOCs in he same g oup. 33 In his wo k, he ege ables ed peppe and g een peppe , ollowed by oma o, we e he majo oods con ibu ing o i amin C in ake in ou sample. In a p e ious s udy by o he au ho s, high NOC concen a ions we e associa ed wi h longe ansi ime and lowe ecal weigh , sugges ing mo e e icien mic obio a media ed o ma ion and accumula ion o hese compounds in eces. 51 In con as , a highe in ake o die a y ibe can inc ease s ool olume and sho en ansi ime, leading o lowe NOC concen a ions and dec easing hei in e ac ions wi h he colon mucosa. In his s udy, i was no possible o calcula e daily exc e ion o NOCs. The e o e, he concen a ions epo ed he e could be in luenced by con ounding ac o s, such as ecal olume and ansi ime. Al hough wi h hese da a we canno es ablish causali y be ween he in ake o ed and p ocessed mea s, ni a es, ni i es, and NAs de i ed om ood p ocessing wi h he endogenous o ma ion o NOCs, i seems clea om ou esul s ha hese ac o s a e associa ed. The e o e, he nex ques ion is whe he his inc eased exc e ion o NOCs is associa ed wi h inc eased ecal oxici y. Some p e ious wo k add essing his issue had ound an inc ease in ecal wa e geno oxici y a e ed mea consump ion du ing an in e en ion s udy al hough non- signi ican inc emen s o NOC ecal le els we e obse ed. 52 In ou s udy, he g oup o high ecal mu agenici y showed he highes o al and heme ecal NOC concen a ions, and acco ding o he his opa hology g oup, s ong associa ions be ween ecal heme NOCs and ecal mu agenici y we e no able only in he case o he HP g oup. In he p esen wo k, no di e ences we e ound in he in ake o ni i es and die a y NOCs be ween he his opa hology g oups, bu we obse ed ha hese compounds we e mainly de i ed om he consump ion o p ocessed mea s and we e posi i ely co ela ed wi h ecal o al and heme NOC concen a ions. Inc eased ecal NOC concen a ions we e no ed among indi iduals consuming highe amoun s o p ocessed mea han ecommended by egula o y agencies as well as in associa ion wi h he inc ease in he se e i y o colonic mucosal damage om NP o AC, and in samples wi h highe ecal mu agenici y. The s udy o he associa ions among die a y componen s and endogenous NOCs conside ing he di e en in es inal en i on- men s may help o unde s and hei impac on colonic mucosal damage and he p og ession o ce ain diseases, such as CRC. ■AUTHOR INFORMATION Co esponding Au ho Sonia González −Depa men o Func ional Biology, Uni e si y o O iedo, 33006 O iedo, Spain; Die , Mic obio a and Heal h G oup, Ins i u o de In es igación Sani a ia del P incipado de As u ias (ISPA), 33011 O iedo, Spain; o cid.o g/0000- 0003-2602-7036; Email: [email p o ec ed] Au ho s Se gio Ruiz-Saa ed a −Depa men o Mic obiology and Biochemis y o Dai y P oduc s, Ins i u o de P oduc os Lác eos de As u ias (IPLA-CSIC), 33300 Villa iciosa, Spain; Die , Mic obio a and Heal h G oup, Ins i u o de In es igación Sani a ia del P incipado de As u ias (ISPA), 33011 O iedo, Spain Tuulia K ee a Pie ilä −Depa men o Food and Nu i ion, Uni e si y o Helsinki, 00014 Helsinki, Finland Aida Zapico −Depa men o Func ional Biology, Uni e si y o O iedo, 33006 O iedo, Spain; Die , Mic obio a and Heal h G oup, Ins i u o de In es igación Sani a ia del P incipado de As u ias (ISPA), 33011 O iedo, Spain Cla a G. de los Reyes-Ga ilán −Depa men o Mic obiology and Biochemis y o Dai y P oduc s, Ins i u o de P oduc os Lác eos de As u ias (IPLA-CSIC), 33300 Villa iciosa, Spain; Die , Mic obio a and Heal h G oup, Ins i u o de In es igación Sani a ia del P incipado de As u ias (ISPA), 33011 O iedo, Spain Anne-Ma ia Paja i −Depa men o Food and Nu i ion, Uni e si y o Helsinki, 00014 Helsinki, Finland Comple e con ac in o ma ion is a ailable a : h ps://pubs.acs.o g/10.1021/acs.ja c.4c05751 Funding This s udy ecei ed inancial suppo om he RTI2018- 098288-B-I00 p ojec (MIXED) inanced by MCIN/AEI/ 10.13039/501100011033 and by FEDER “Una mane a de hace Eu opa”, om PID2022−140410OB-I00 p ojec (MiTox- icDie ) inanced by MCIN/AEI/10.13039/501100011033 and by “ERDF A way o making Eu ope” and om he AYUD/2021/ 50981 p ojec om he P incipali y o As u ias o suppo he ac i i y o esea ch g oups. S.R.-S. andA.Z. we e he ecipien s o a p edoc o al con ac (Se e o Ochoa 2021-BP20−012 and PA- 23-BP22−034, espec i ely) unded by he Plan Regional de In es igacion om he P incipali y o As u ias. No es The au ho s decla e no compe ing inancial in e es . ■ACKNOWLEDGMENTS We a e indeb ed o all pa ien s who ha e pa icipa ed in he s udy and o he nu ses and he acul a i es A.S., C.G.d.R., and Y.D. a he Diges i e Se ices o HUCA and Ca men and Se e o Ochoa Hospi als in As u ias, No he n Spain. ■REFERENCES (1) Wo ld Cance Resea ch Fund In e na ional (WCRF)/Ame ican Ins i u e o Cance Resea ch (AICR). Die , nu i ion, physical ac i i y and colo ec al cance . Con inuous Upda e P ojec Expe Repo ; Wo ld Cance Resea ch Fund In e na ional: London, U.K, 2018; 1−62. (2) Die ich, M.; Block, G.; Pogoda, J. M.; Bu le , P.; Hech , S.; P es on-Ma in, S. A e iew: die a y and endogenously o med N- ni oso compounds and isk o childhood b ain umo s. Cance Causes Con ol. 2005,16, 619−635. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c05751 J. Ag ic. Food Chem. 2024, 72, 17588−17598 17596