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
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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
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h ps://doi.o g/10.1021/acs.ja c.4c05751
J. Ag ic. Food Chem. 2024, 72, 17588−17598
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