Local Acetaldehyde—An Essential Role in Alcohol-Related Upper Gastrointestinal Tract ...

23 Pages·2017·0.71 MB·English

cancers
Review
Local Acetaldehyde—An Essential Role in
Alcohol-Related Upper Gastrointestinal
Tract Carcinogenesis
MikkoT.Nieminen1,2 ID andMikkoSalaspuro2,*
1 DepartmentofOralandMaxillofacialDiseases,UniversityofHelsinki,andHelsinkiUniversityCentral
Hospital,UniversityofHelsinki,BiomedicumHelsinkiP.O.Box63,00014Helsinki,Finland;
mikko.t.nieminen@helsinki.fi
2 ResearchUnitonAcetaldehydeandCancer,UniversityofHelsinki,BiomedicumHelsinkiP.O.Box63,
00014Helsinki,Finland
* Correspondence:mikko.salaspuro@helsinki.fi;Tel.:[phone]
Received:23November2017;Accepted:20December2017;Published:5January2018
Abstract: Theresidentmicrobiomeplaysakeyroleinexposureoftheuppergastrointestinal(GI)
tract mucosa to acetaldehyde (ACH), a carcinogenic metabolite of ethanol. Poor oral health is a
significant risk factor for oral and esophageal carcinogenesis and is characterized by a dysbiotic
microbiome. Dysbiosisleadstoincreasedgrowthofopportunisticpathogens(suchasCandidayeasts)
andmaycauseanupto100%increaseinthelocalACHproduction,whichisfurthermodifiedby
organ-specificexpressionandgenepolymorphismsofethanol-metabolizingandACH-metabolizing
enzymes.Apointmutationinthealdehydedehydrogenase2genehasrandomizedmillionsofalcohol
consumerstomarkedlyincreasedlocalACHexposureviasalivaandgastricjuice,whichisassociated
withamanifoldriskforupperGItractcancers. Thishumancancermodelprovesconclusivelythe
causalrelationshipbetweenACHandupperGItractcarcinogenesisandprovidesnovelpossibilities
forthequantitativeassessmentofACHcarcinogenicityinthehumanoropharynx. ACHformedfrom
ethanolpresentin“non-alcoholic”beverages,fermentedfood,oraddedduringfoodpreparation
formsasignificantepidemiologicbiasincancerepidemiology. Thesamealsoconcerns“free”ACH
presentinmutagenicconcentrationsinmultiplebeveragesandfoodstuffs. LocalexposuretoACH
iscumulativeandcanbereducedmarkedlybothatthepopulationandindividuallevel. Atbest,
apersonwouldneverconsumetobacco,alcohol,orboth. However,evensmokingcessationand
moderationofalcoholconsumptionareassociatedwithamarkeddecreaseinlocalACHexposure
andcancerrisk,especiallyamongestablishedriskgroups.
Keywords: acetaldehyde;ADH;alcohol;ALDH;ALDH2;cancer;ethanol;fermentedfood;tobacco;
uppergastrointestinaltract
1. Introduction
Alcohol is a major risk factor for upper gastrointestinal (GI) tract cancer and exhibits a
dose-dependenteffectontheincidenceoforopharyngealandesophagealsquamouscellcancers[1].
Asimilarbutweakerdose-riskrelationshipalsoexistswithalcohol-relatedgastriccancer[1,2]. There
isnoevidencethattheethanolmoleculeitselfisgenotoxic,mutagenic,orcarcinogenic[3,4]. However,
itsfirstmetabolite, acetaldehyde(ACH),isagroup1carcinogentohumanswhenassociatedwith
consumptionofalcoholicbeverages[5]. TheInternationalAgencyforResearchonCancer(IARC’s)
conclusionconcernsmetabolicallyformedACHfromethanoland“free”ACHpresentinalcoholic
beverages[5,6]. Therefore,knowledgeofallfactorsregulatingACHconcentrationintheupperGI
Cancers 2018,10,11;doi:10.3390/cancers10010011 www.mdpi.com/journal/cancers
Cancers 2018,10,11 2of23
tractisofessentialimportanceforcancerprevention. Inotheralcohol-relatedcancers,suchasliverand
femalebreastcancers,thecarcinogenicmechanismsaredifferentandbyandlargestillhypothetical.
Apointmutationinthealdehydedehydrogenase2(ALDH2)generesultsindeficientactivity
ofthemainACH-metabolizingmitochondrialenzyme(ALDH2)andprovesconclusivelythecausal
relationshipbetweenlocalACHexposureandupperGItractcancers(Table1;[7,8]). Bacteriaand
yeastspresentinthenormalupperGItractmicrobiomeplayamajorroleinlocalACHformation
fromethanolpresenteitherinalcoholicbeveragesorfood. Withinseconds,alcoholingestionresults
in a dose-dependent accumulation of ACH in saliva. The prominent instant increase of salivary
ACH concentration is followed by a long-term phase, which lasts for as long as ethanol stays in
thehumanbody. HostmetabolismofethanolandACHarecontrolledbyorgan-specificexpression
andgenepolymorphismsofethanol-metabolizingandACH-metabolizingenzymesandcontribute
to microbial ACH formation. The most important exogenous sources for local ACH exposure are
alcoholicbeverages, tobacco, andalcohol-containingorACH-containing(orboth)“non-alcoholic”
beveragesandfood.
Table1.Maincharacteristicsofthegenemutation-basedhumanmodelforthequantitativeassessment
of acetaldehyde (ACH) carcinogenicity in alcohol-consuming aldehyde dehydrogenase (ALDH)
2-deficientsubjectscomparedtoALDH2-activesubjects[7,8].
(cid:226) Theethanolmoleculeisneithergenotoxic,mutagenic,norcarcinogenic
(cid:226) ACHassociatedwithalcoholconsumptionisaGroup1humancarcinogen
(cid:226) ACHdoesnotexistinsalivawithoutthepresenceofethanolortobacco
(cid:226) ACHaccumulatesafteralcoholintakeinabout2timeshigherconcentrationsinsalivain
ALDH2-deficientsthaninALDH2-actives
(cid:226) Theoropharynxandesophagus(?)asidealtargetorgansforquantificationofACH-relatedcancerrisk
inhumans
(cid:4) Insharpcontrasttotheliver,theoralmucosalackslow-KMALDHenzymesandisthusunableto
eliminateacetaldehydeformedfromethanol
(cid:4) TheexpressionofALDHenzymesislowintheesophagus,buttheirroleintheregulationoflocal
ACHlevelsisstillunknown
(cid:4) TheoralmicroflorahasloworzerocapacitytoeliminateACH
(cid:4) AdequatedataonsalivaryACHinALDH2-deficientsubjectsvs.ALDH2-activesubjects;
equivalentdataonACHofthemucosalsurfaceoftheesophagusisstillunavailable
(cid:4) AdequateepidemiologicaldataontheroleofALDH2polymorphismsontheriskfor
oropharyngealcanceramongnever,moderate,andheavydrinkers
(cid:226) NodifferencesbetweenALDH2-deficientsubjectsandALDH2-activesubjectsinconfoundingfactors
hamperingmostepidemiologicalstudiesonalcohol-relatedcancer
(cid:4) Smoking,diet,consumptionofdifferentbeverages,varyingdrinkinghabits,underreporting,oral
hygienelevels,humanpapillomavirus(HPV),bodymassindex(BMI)
2. Acetaldehyde
2.1. GenotoxicityandMutagenicity
InvitroandinvivogenotoxicityofACHisgenerallyaccepted[3,5,9]. ACHfulfillsfourofthe
tenkeycharacteristicsthatarecommonlyexhibitedbyestablishedhumancarcinogens[10]. ACH
iselectrophilicandgenotoxic,altersDNArepair,andinducesoxidativestress. Whenadministered
inrelativelyhighconcentrationsinvitro,ACHcausesDNA-proteincrosslinks,DNA-strandbreaks,
DNAadducts,sisterchromatidexchanges,chromosomalaberrations,andmicronucleiineukaryotic
cells. In a study using pig liver DNA, ACH concentrations ranging from 100 to 500 µM in
the presence of polyamines resulted in an exponential increase of mutagenic ACH-DNA adduct
(1,N2-propanodeoxyguanosine)formation[11]. TheseACHlevelsarerelevantforprevailingsalivary
Cancers 2018,10,11 3of23
ACHconcentrationsafteralcoholingestion[12,13]. Ontheotherhand,increasedpolyaminesynthesis
is characteristic to regenerating oral and esophageal mucosa [14]. In 2015, the UK Committee on
MutagenicityconcludedthattheweightofevidencefortheinvitromutagenicityofACHhasbeen
furtherstrengthened,particularlywithregardtogenerationofspecificDNAadductsandinductionof
micronucleiinmammaliancellsatACHconcentrationsrealisticallyachievablefromconsumptionof
alcoholicbeverages[3].
Alcohol-treatedALDH2-knockoutmiceshowincreasedlevelsofmutagenicACH-DNAadducts
inthetongue,submandibularglands,esophagus,stomachandliver[15–17]. Furthermore,Japanese
alcoholics deficient in the ability to detoxify ACH (ALDH2 deficiency) combined with enhanced
abilitytoproduceACHfromethanol(highlyactivealcoholdehydrogenase[ADH]enzyme)show
markedlyenhancedDNAdamagecharacterizedbyelevatedlevelsofN2-ethylidene-dGadductsin
bloodDNA[18].
A 12-fold to 16-fold increase in mutagenic ACH-DNA adducts (N2-ethylidene-dG) has been
demonstratedinmouthwashsamplesofhealthyhumanvolunteersat2and4hafteringestionofa
moderatedoseofalcoholaimedtoreach0.7 bloodalcoholconcentration[19]. Inanalcoholsipping
model (diluted vodka at 5 min intervals) u(cid:104)sed in that study, salivary ACH concentration can be
assumedtohavebeenapproximately150µM(maximum260µM)forthefirst20to40minfollowed
byarapiddecreaseto20to30µMfortheremainderofthefollow-upperiod(350min)[8]. Mutagenic
ACHconcentrationhasbeenestimatedtorangefrom40to100µM[20].
TheformationofmutagenicACH-DNAadductsinoralmucosahasbeenconfirmedinrhesus
monkeysexposedtoalcoholovertheirlifetimes[21]. Incontrasttothesefindings,alcoholdrinking
didnotappeartoinducesignificantlyincreasedlevelsofN2-ethylidene-dGadductsinbloodwhite
cellsofhumanvolunteers[22,23]. Thisdiscrepancy,however,isnotsurprising,sinceonlyverylow
ACHconcentrationsarefoundinhepaticvenousbloodofintoxicatednon-alcoholicmaleCaucasians,
andACHlevelsareundetectable(<2µM)inperipheralbloodsamples[24,25].
2.2. CarcinogenicityinExperimentalAnimals
There is sufficient evidence for the carcinogenicity of ACH in experimental animals [26].
Ininhalationtoxicitystudieswithrats,ACHvaporforupto28monthsproducedadenocarcinomasof
theolfactoryepitheliumatallexposureconcentrationsandsquamouscellcarcinomasoftherespiratory
epitheliumatthetwohighestACHlevels[27,28]. ExposuretoACHvaporfor52weeksproduced
nasalandlaryngealcarcinomasinSyriangoldenhamsters[29]. Withoutanobviousdose-response
relationship,lifetimeadministrationofACHindrinkingwatertoratsresultedinincreasednumber
of pancreatic adenomas, lymphomas, leukemias, uterine and mammary gland adenocarcinomas,
andheadosteosarcomas[30].
2.3. CarcinogenicityinHumans
CasereportsandepidemiologicalstudiesonthecarcinogenicityofACHinhumanswithoutthe
presenceofethanolareverylimited. Ninecasesofmalignantneoplasmswerereportedamongan
unspecifiednumberofworkersinanaldehydefactoryintheGermanDemocraticRepublicbetween
1967and1972[31]. Ofthecancersfound,fivewerebronchialtumorsandtwowerecarcinomasof
theoralcavity. Theauthorsstatedthattherelativefrequenciesofthesetumorswerehigherthanthe
expectedfrequencyintheGermanDemocraticRepublic.
However, present epidemiologic and biochemical evidence on ALDH2-deficient individuals
providesauniqueandquantifiableACHexposuremodelinhumans,thekindofwhichisnotavailable
foranyotherofthegroup1carcinogens(Table1,[7,8]). Naturehas“randomized”millionsofalcohol
consumerstomarkedlyelevatedlocalACHconcentrationsviasalivaeverytimewhendrinkingalcohol.
TheenhancedACHexposureviasalivacontinuesforaslongasalcoholstaysinbloodcirculation. Both
ofthesefactorscanbequantifiedandlinkedtotheriskfororopharyngealcancerinrelationtothe
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amountofalcoholconsumeddaily. Mostimportantly,themodelisnotbiasedbyconfoundingfactors
thathampermostepidemiologicalstudiesonalcohol-relatedcancer.
2.3.1. ALDH2-DeficiencyasaUniqueHumanCancerModel
AsinglepointmutationinALDH2generesultsindeficientactivityofthemainACHmetabolizing
lowK mitochondrialenzyme(ALDH2). Consequently,ALDH2-deficientsubjectsareexposedinthe
M
presenceofalcoholtwotothreetimes(viasaliva)andfivetosixtimes(viagastricjuice)higherACH
concentrationsthanthosewiththeactiveALDH2enzyme[32–36]. ParalleltoincreasedlocalACH
exposure,theriskofALDH2-deficientalcoholdrinkersfororal,pharyngeal,esophageal,andgastric
cancerismanifoldcomparedtoalcohol-consumingindividualswiththeactiveALDH2-enzyme[37–43].
Themutatedgene(ALDH2*487Lysallele)isderivedfromtheancientPai-Yueitribe,whichwaswidely
distributed along the southeast coast of China up to Yunnan Province and the northern part of
SoutheastAsia2000–3000yearsago[44]. Todayitscarrierfrequencyisabout600millionpeopleof
EasternAsiandescent[44,45]. Thus,ALDH2-deficiencyisthemostprevalentgenetichealthriskin
theworld,passinginfrequencytothatoffamilialhypercholesterolemia(FH).TheprevalenceofFHis
1:200–250,butincontrastALDH2deficiencyis1:13[44–46].
2.3.2. QuantitativeAssessmentoftheCarcinogenicityofAcetaldehydeinHumans
TheriskassessmentforACHhasthusfarreliedonthresholdsbasedonanimaltoxicologywith
lowerone-sidedconfidencelimitofthebenchmarkdosevalues(BMDL),typicallyrangingbetween11
and63mg/kgbodyweight(bw)/daydependingonspeciesandendpoint[7].However,theanimaldata
isproblematicduetopoorstudyquality,problemsinanimalmodels,andrelevanceoftheendpoint
(cancerlinkage)intranslationtohumans[7]. AccordingtoBMDLmodeling,ALDH2-deficientheavy
drinkers can be calculated to be exposed to a mean of 6.7 µg/kg bw/day of additional ACH via
salivacomparedtoALDH2-activeindividuals[7]. Thisassociateswithoddsratiosofuptosevenfor
esophagealandheadandneckcancer[7]. ThesenumbersderivedfromanALDH2-deficienthuman
cancermodelsuggestmarkedlylowerBMDLvaluesforACH(<0.1mg/kgbw/day)thantheones
calculatedfromstudiesonexperimentalanimals(11–63mg/kgbw/day). Unfortunately,however,
human data does not allow for a dose-response-modeling as none of the epidemiological studies
provideabsoluteorextrariskdata[7].
Asimplerwayistocalculateareaunderthecurve(AUC)oflocalACHexposurebymultiplying
salivary ACH concentration with exposure time (Table 2). Based on five studies with uniform
results,salivaryACHconcentrationisamean1.1mg/LhigherinALDH2-deficientsubjectsthanin
ALDH2-activesubjectsforaslongasalcoholstaysinthehumanbodyafteritsintake[7]. Onthebasis
ofthealcoholeliminationrate(7g/h),theincreasedexposuretimetosalivaryACHcausedbythe
deficientALDH2enzymeis283mininmoderatedrinkersand660mininheavydrinkers.Thetotal
additionalexposureoftheoropharynxtoACHcausedbyALDH2-deficiencyisthus311mg/L/283min
inmoderatedrinkersand726mg/L/660mininheavydrinkers. Accordingtoonemeta-analysisand
onewell-performedstudy(totalof1189casesand3239controls),ALDH2deficiencyisassociatedwith
a1.68-foldto2.61-foldoropharyngealcancerriskinmoderatedrinkersand3.57-foldto7.28-foldriskin
heavydrinkers[37,39]. TheoropharyngealcancerriskofALDH2-deficientalcoholdrinkerscompared
withthatofALDH2-activeindividualsthusappearstohaveadose-dependentcorrelationwiththe
additionallocalACHexposurecausedbythedeficientALDH2enzyme[8].
Cancers 2018,10,11 5of23
Table2. EffectofALDH2deficiency,alcoholconsumption,smoking,andcertainfoodstuffsonthe
quantitative exposure of oropharynx to acetaldehyde (ACH) via saliva in relation to the risk for
oropharyngealcancer.Calculationsarebasedonselectedexamplesasindicatedinfooters.
SalivaryACH OR/RRfor
ExposureTime ACHExposure
ExposureModel Concentration Oropharyngeal
(min) (mg/L×min)/day
(mg/L;µM) Cancer
ALDH2-DeficiencyModel
-3doses(33gethanol)/day 1.1;251 2832 3111 1.68–2.613
-7doses(77gethanol)/day 1.1;251 6602 7261 3.57–7.283
1Dose40%Alcohol,noACH4
-Instant
3×first5min 6.2;150 15 93
1×next5min 4.4;100 5 22
instanttotal 115 1.295
-Long-term
20–80min 0.88;20 60 53
-Total
0–80min 80 168
Smoking6
-1cigarette 11.5;261 5 58
-3–5cigarettes 11.5;261 15–25 173–288 2.017
Yogurt
-Safest-casescenario8 2.4;55 3 7 ?
-Worst-casescenario9 17.4;395 3×5 261
Apple
-Safest-casescenario10 0.3;7 3 0.9 ?
-Worst-casescenario11 2.4;55 3x5 36
1 Difference in salivary ACH concentration (column 2) or in ACH exposure (column 4) (ALDH2-deficient
subjects—ALDH2-activesubjects)[7]; 2 Basedonthenormaleliminationrateofethanol(7g/h)[47]; 3 Odds
ratios(ORs)fororopharyngealcancer, ALDH2-deficientsubjectscomparedtoALDH2-activesubjects[37,39];
4 Onedoseofalcoholisassumedtobeingestedinthreesipsat5-minuteintervalsstayingeachinthemouth
forabout5sbeforeswallowing. Adaptedfrom[12,13,48]; 5 Relativerisk(RR)oforopharyngealcancerfor1
dose alcohol (10g ethanol)/day [49]; 6 Adapted from [50]; 7 OR for head and neck cancer in never drinkers
smoking > 3–5 cigarettes/day [51]; 8 One commercially available yogurt (approximately 150 mL)/day with
lowestreportedACHlevel, consumedin3min. Adaptedfrom[52]; 9 Threecommerciallyavailableyogurts
(approximately3×150mL)/daywithhighestreportedACHlevel,eachconsumedin5min.Adaptedfrom[52];
10Onecommerciallyavailableapple/daywithlowestreportedACHlevel,consumedin3min.Adaptedfrom[52];
11Threecommerciallyavailableapples/daywithhighestreportedACHlevel,eachconsumedin5min.Adapted
from[52].?:nodataavailable.
Heavyalcoholdrinking(ethanol≥50g/day)comparedtonon-drinkersandoccasionaldrinkers
is associated with a 5.13-fold risk for oropharyngeal cancer [1]. From 50 g of ethanol, the human
body produces mathematically over 1 × 106 µmoles of ACH. Hepatic ALDH enzymes, however,
oxidize ACH formed from ethanol so effectively that after a moderate to high dose of alcohol
(0.4–1.2g/kg)bloodACHlevelsarenotdetectableinhealthyindividualswiththenormalALDH2
enzyme [24,25,53]. In ALDH2-deficient individuals, blood ACH levels are only slightly elevated
(≤10 µM) [33,34,36]. In sharp contrast to the liver, the oral mucosa lacks low K ALDH enzymes
M
andisthusunabletoeliminateACHformedfromethanolintheoralcavity[54]. Thecapacityoforal
microbestoeliminateACHisalsolow,andinthisrespect,thereisnoevidenceforanydifferences
betweenALDH2-deficientandALDH2-activeindividuals[55]. Therefore,alcoholdrinkingresults
in the accumulation of mutagenic concentrations of ACH in the saliva of both ALDH2-active and
ALDH2-deficientindividualsforaslongasethanolstaysinthehumanbody[12,13,33–36].
Cancers 2018,10,11 6of23
2.3.3. ImplicationsoftheALDH2CancerModelforRegulatoryAuthorities
There is no evidence that ACH derived from ethanol present sometimes in percentage
concentrationsin“non-alcoholic”beveragesorfoodislesscarcinogenictotheupperGItractmucosa
thanACHformedmetabolicallyfromtheethanolofofficialalcoholicbeverages. Thisalsoconcerns
ACH found in mutagenic concentrations in fermented foodstuffs or when present in the product
as a natural or added flavor [52,56]. There are no safe limits for very high (up to >1800 mg/L to
>45,000µM)ACHconcentrationsmeasuredinsomealcoholicbeverages[57,58]. Thiscanbeexplained
atleastinpartduetotheclassificationofACHasa“generallyrecognizedassafe”(GRAS)substrate
validatedbytheJointFoodandAgricultureOrganizationoftheUnitedNations(FAO)andWorld
HealthOrganization(WHO)ExpertCommitteeonFoodAdditives(JECFA)in1998andtheJapan
FlavourandFragranceMaterialsAssociation(JFFMA)in2015[59,60].Inappropriately,thedecisiontree
forthesafetyassessmentofflavoringsubstancesappliedbyJECFA,JFFMA,theFlavourandExtract
ManufacturesAssociation(FEMA)ExpertPanel,andtheEuropeanFoodSafetyAuthority(EFSA)is
basedonamisconceptionconcerningACHmetabolism. TheWHOmodelusedbytheseauthorities
predictsthatACHundergoescompletemetabolismtosafeproducts[59,61].Thisassumption,however,
doesnotconcernACH,whichaccumulatesinmutagenicconcentrationsinsalivainthepresenceofeven
verylowethanolconcentrations(10mM,0.5 [12,13]. Furthermore,apeak(upto1000µM)insalivary
ACHisseeninstantlyaftersippingofalcoh(cid:104)olicbeveragescontaininghighlevelsofACH[13,36,62].
Genotoxic substances are generally not permitted for deliberate use in food production,
since there is an increased risk for cancer even at low exposure levels. Therefore, controlling
exposurelevelstoALARA(EFSA)orALARP(UK)(AsLowasReasonablyAchievableorAsLow
as Reasonably Practicable, respectively) are generally accepted principles concerning genotoxic,
mutagenic,andcarcinogenicsubstances[10,63–65]. ThescientificcommitteeofEFSAhasidentified
withinitsprioritytasksthedevelopmentofjustifiableapproachesfortheassessmentofrisksfrom
substancesthatarebothgenotoxicandcarcinogenic[65]. Tothataim,theALDH2-deficiencymodelfor
localACHexposureinthehumanoropharynxprovidesanoveltoolforthequantitativereevaluation
ofACHcarcinogenicityinhumans[7,8]. Akeyprerequisiteforthatapproachwillbethattheamounts
ofethanolandACHinbeveragesandfoodstuffsaremadegenerallyavailableforresearchers,health
careworkers,regulatoryauthorities,andconsumers.
3. MicrobialMetabolism—AMajorSourceofLocalAcetaldehydeintheUpperGITract
3.1. SalivaryAcetaldehyde—InstantandLong-TermExposure
MultiplefactorscontributetosalivaryACHlevels(Figure1). Normalhumansalivadoesnot
containmeasurablelevelsofACHwithoutthepresenceofethanolortobacco[12,50]. Aftersippinga
smalldose(5mL)of40%alcohol,ethanolisdistributedtooralmucosalsurfacesandsalivarapidly,
resultinginanupto900mM(4.4%)salivaryethanolconcentrationat30s[13,48]. Instantmicrobial
ACHformationfromethanolstartssubsequentlyandcontinuesfor5to10minaftereachsipofalcohol
(Table2;[13]). CertainmembersofthenormaloralmicroflorapossessparticularlyhighADHactivity
andareabletodose-dependentlyproduceACHinvivo/invitroinincreasingethanolconcentrations
from11to1500mM(0.5 –7%)[12,66,67]. ThisindicatesratherhighK valuesforspecificmicrobial
M
ADHsandexplainswhy(cid:104)invivosalivaryACHconcentrationsarehighest(upto260µM)immediately
aftersippingofalcohol[13].
Onedoseofalcohol(10gethanol)dailyisassociatedwithasignificantrelativerisk(RR)of1.17
(approximately20%increase)fororopharyngealcancer[49,68]. Alcoholintakeinthreesipsat5-min
intervalsisestimatedtoresultininstant(meanapproximately115mg/L/20min)exposureofthe
oropharynxtoACH,whichrepresentscloseto70%ofthetotal(168mg/L/80min)ACHexposure
(Table2,[13]). Long-termexposure(inthisexampleapproximately53mg/L/60min)consistsofACH
formedfromethanolthatisdiffusedintosalivafrombloodandlastsforaslongasethanolispresent
in the human body [12]. High concentrations of “free” ACH present in some alcoholic beverages
Cancers 2018,10,11 7of23
hasbeenlinkedtotheparticularlyhighincidenceofalcohol-relatedupperGItractcancers[69–71].
Thishypothesisissupportedbybiochemicalfindingsshowingthatsippingofalcoholicbeverages
containing“free”ACHfrom470µMtoover15.5mMresultsinashort,1to2minpeak(upto≥1mM)
insalivaryACHconcentration[13,36,62]. Drinkinghabits,suchasnumberofsips/doseandhighACH
Cancers 2018, 10, 11 7 of 23
concentrationinthealcoholicbeverageconsumedmaythushaveasignificanteffectontheexposure
ACH concentration in the alcoholic beverage consumed may thus have a significant effect on the
oforopharyngealmucosatoACH.
exposure of oropharyngeal mucosa to ACH.
Alcohol spirits / beverages
Dietary sources
Tobacco
products
Other
Sugars Ethanol
O ↓ ADH O ↑
2 2 Salivary
Acetaldehyde
Pyruvate Acetaldehyde
Acetate Molecular damage
Microbial
metabolism Mutagenesis
Host
metabolism
Oropharyngeal
carcinogenesis

Figure 1. Schematic overview of the major factors contributing to salivary acetaldehyde (ACH) levels
Figure1.Schematicoverviewofthemajorfactorscontributingtosalivaryacetaldehyde(ACH)levels
and therefore the local ACH exposure of oropharyngeal mucosa. ACH exposure leads to molecular
andthereforethelocalACHexposureoforopharyngealmucosa.ACHexposureleadstomolecular
changes and mutagenesis, including DNA-adduct formation, DNA-protein crosslinks, DNA strand
changesandbremakus taangde cnhreosmiso,sionmcallu adbeinrrgatiDonNs. ALo-caadllyd, uthcetsef ochrmangaetsi omna,yD leNadA to- pdyrosptleaisnia carnods fsulritnhekrs i,nDto NAstrand
breaksandocrhoprohamryonsgoeaml caalncaebr.e Mrricartoiboinals a.nLd ohcoastl mlye,ttahboelsisemc shhaanreg thees ambilaityy ltoe acodnvtoertd eythsapnloal stoia AaCnHd byfu rtherinto
the alcohol dehydrogenase enzyme (ADH). There is plethora of microbes that possess highly-active
oropharyngealcancer.MicrobialandhostmetabolismsharetheabilitytoconvertethanoltoACHby
ADH enzymes. However, the subsequent conversion of ACH to less harmful substances seems to be
thealcoholvdeeryh lyimdirtoedg deunea tsoe theen lzayckm oef a(cAtivDe Hal)d.eThyhdeer deeihsydprloegtehnoasrea (AofLDmHic) reonzbyemsetsh ina tbpotohs mseicsrsobhiailg hly-active
ADHenzymaneds .orHopohwareynvgeera,lt mheucsousabl sceelqlsu. eInn ctocnotrnavste tros tihoen houmfAanC hHostt, oetlheasnsolh faerrmmefnutaltisoun biss taa snpceecisfics eemstobe
verylimitedtrdaiut feort omtichreobleasc. kIno lofwa cotxiyvgeena ltdeneshioynd, esudgaerhs ydderriovegde nfraomse d(iAetLarDy Hsou)recnesz, ysumche sasi nglubcootshe amndic robialand
fructose, are converted into ethanol and ACH is formed as a byproduct. The possible contribution of
oropharyngealmucosalcells.Incontrasttothehumanhost,ethanolfermentationisaspecifictraitfor
fermentative pathways from glucose to local ACH production and further to ethanol has not been
microbes.Inexlpolworeodx tyhugse fnart ienn vsiivoon. I,ns audgdaitriosnd teo railvcoehdolfirco bmevedraiegetas raynds ospuirrictse, sm,usluticphle adsiegtalruyc coosnestiatunedntfsr uctose,are
convertedincatno ceotnhtaainn eotlhaannodl, AACCHH, oris bofothr.m Theedre aasrea albsoy optrhoerd suouctr.ceTs hofe epthoanssoil,b sluechc oasn htryigbieuntei oprnodoufcftse,r mentative
including daily oral mouthwash use. In addition to alcohol, tobacco is the major contributor to
pathwaysfromglucosetolocalACHproductionandfurthertoethanolhasnotbeenexploredthus
salivary ACH.
farinvivo. Inadditiontoalcoholicbeveragesandspirits,multipledietaryconstituentscancontain
ethanol3,.2A. COrHal, aonrd bEosotphh.aTgehale Mreicarroebioamlseo othersourcesofethanol,suchashygieneproducts,including
dailyoralmAosu htihgwhlaigshhteuds eea.rIlniera, dsadliivtiaorny AtoCaHlc ios hmoali,ntloyb oaf cmcoicriosbtihael omrigaijno.r Tchoenretr iisb au sttorrontog csoarlrivelaartiyonA CH.
between in vivo and in vitro salivary ACH production [12]. 4-methylpyrazole (4-MP) is an effective
3.2. OralandcoEmspoeptihtiavge eianlhMibiitcorro obfi otmhee human ADH enzyme. Approximately 10 to 20 times higher 4-MP
concentrations are necessary for 40 to 50% inhibition of in vitro microbial ACH production from
Ashigehthliagnholt eind saelaivral iaenrd, osraalli vbaacrteyriAal ClyHsateiss tmhaani fnorly eqoufalm inihcirboitbioina olfo ertihganinol. eTlihmeinraetiiosna ins htruomnagnsc orrelation
betweenin[v12iv,3o4,6a7n]. dIni nALvDitHro2-ascatilviev asurbyjeActsC, H4-MpPr o(1d0–u1c5t miogn/k[g1) 2d]i.d4 n-omt reetdhuycelp syalrivaazroy lAeC(H4- MlevPel)s ibsuat neffective
decreased the ethanol elimination rate by 46% [34]. This indicates the minor role of mucosal and
competitive inhibitor of the human ADH enzyme. Approximately 10 to 20 times higher 4-MP
glandular ADH and the major role of microbial metabolism in the regulation of salivary ACH
concentratioconnscaenrterantieocne sasfaterry afolcroh4o0l toin5ta0k%e. iFnuhrithbeirtmioonreo, ftihne vuitsreo omf icchrloobrhieaxlidAinCeH (CpHroXd) uacnttiiosenptfirco methanol
insalivaandmoourtahlwbaashc tfeorr itahlreley dsaaytse sbetfhoraen inftoakree oqfu a amloidnehraibtei tdioosne ooff aelctohhaonl doelcerelaimsedin thaet iloevnelisn ofh sualmivaarnys [12,34,67].
ACH significantly (approximately 50%) in comparison with subjects that did not use CHX [12].
InALDH2-activesubjects,4-MP(10–15mg/kg)didnotreducesalivaryACHlevelsbutdecreasedthe
ethanolelim inationrateby46%[34]. ThisindicatestheminorroleofmucosalandglandularADHand
themajorroleofmicrobialmetabolismintheregulationofsalivaryACHconcentrationafteralcohol
intake. Furthermore,theuseofchlorhexidine(CHX)antisepticmouthwashforthreedaysbeforeintake
ofamoderatedoseofalcoholdecreasedthelevelsofsalivaryACHsignificantly(approximately50%)
incomparisonwithsubjectsthatdidnotuseCHX[12].
Cancers 2018,10,11 8of23
3.2.1. OralandEsophagealMicrobiomeinHealthandDisease
PeaklevelsofACHinsalivaafteralcoholingestioncanvarygreatlybetweenindividuals(range
18–260µM;[12,13]). Thesevariationsareduetocompositionalalterationsintheoralmicrobiomeand
levelsofethanolinsalivaafteralcoholingestion. Intheoralcavity,theinterindividualvariationsin
microbialfloramainlyoccuratthespeciesorstrainlevel[72]. Therearedifferentintraoralhabitats
(e.g.,lateral,dorsaltongue,andthegingivalpockets)characterizedbyvariousenvironmentalvariables.
Factors such as oxygen levels, nutrients, and type of surface structure (including squamous cell
epithelium and the enamel of the tooth) affect the microbial community structure and functional
outcomes. Aselucidatedbymultiplemicrobiomestudies,thereisahighlydiverseoralmicrobiome
inhealth,includingover700bacterialspecies[73]. Thebacterialcommunitystructureisdominated
byfivephyla,includingFirmicutes,Proteobacteria,Bacteroidetes,Actinobacteria,andFusobacteria.
Streptococcusistheleadinggenusinthebacterialspectrum,followedbyPrevotella,Veillonella,Neisseria,
andHaemophilus. Theesophagealmicrobiomeisareflectionoftheoralmicrobialflora,broadlysimilar
incompositiontotheoralmicrobiome[74]. Unfortunately,therichfungalflorathatinteractswith
bacteriaintheupperGItractisoftenoverlookedinmicrobiomestudies. Aseukaryotes,fungidiffer
inmultiplewaysfromprokaryoticbacteria. Fungalcellsarelargerinsizeandhavespecificcell-wall
structuresandadvancedmetabolictraits. Themostcommonandwell-studiedaretheCandidayeasts,
especiallyCandidaalbicansspecies. Therearealsootherfungalmembers,suchasAspergillus,Malassezia,
andRhodotorula,buttheirrolesarewidelyunknown.
Poor oral health drives dysbiosis of the microbiome and is associated with dysplasia and
carcinogenesis in the upper GI tract [75–77]. Microbiota isolated from premalignant and oral
carcinomasiteshaveshownmarkedproductionofACHinvitro,thusprovidingsupportforlocalACH
exposure[78]. Insubjectswithpoororalhealth,two-foldhigherinvitrosalivaryACHproduction
from ethanol can be detected compared to individuals with good oral hygiene [79]. Neisseria and
Streptococcusspeciesnotonlydominatethebacterialmicrobiomeinhealthbutalsoindysbiosis[80].
NeisseriaandStreptococcusspeciescandose-dependentlyproducesignificantandmutageniclevelsof
ACHinvitrofromethanol[66,67]. Also,otherspeciesbelongingtothecoreoralmicrobiome(such
as Rothia and Prevotella) are able to produce marked quantities of ACH invitro from ethanol [81].
ThedifferencesbetweenspeciesandgeneraareduetovariationsinbacterialADHenzymeprofiles.
Pavlovaetal. studiedalcoholmetabolisminstreptococciandreportedthatmanystreptococcalspecies
havehighlyactiveADHbutlackanactiveALDHenzyme[55]. Incertainspecies,ADHandALDH
enzymes are in the same enzyme complex. Similarly, no active ALDH enzyme has been found in
Neisseria[67].
3.2.2. CandidaYeasts—AMajorContributortoLocalAlcoholMetabolism
In addition to bacterial counterparts, the fungal flora (Candida genus in particular) is a major
contributortolocalACHexposure. ThepresenceofCandidayeastscorrelatessignificantlywithhigh
salivaryACHlevelsinvitro[82]. Candidayeastsareopportunisticorganismsandthustheirnumber
increasesindysbioticconditions(suchasinindividualswithpoororalhygieneorimpairedimmunity).
BothC.albicansandnon-C.albicansspecies,suchasCandidaglabrataandCandidatropicalis,canproduce
carcinogeniclevelsofACHfromethanolinvitro[83,84]. TosupporttheroleofCandidayeastsinlocal
ethanolmetabolismanditslinkagetomalignanttransformation,C.albicansADHmRNAexpression
hasbeendetectedinoraldysplasiainvivo[85]. Furthermore,thevirulenceattributesofC.albicansand
ACHproductionhavebeenshowntocorrelatewiththepresenceoforalcancer[86]. ChronicCandida
infectionshavebeenlinkedtooralandesophagealcarcinogenesisinsusceptibleindividuals[87,88].
Candidayeastsareknownfortheirfermentativetraits,thusdrivingthemetabolicfluxfromcarbon
sources(suchasglucoseandfructose)intoACHandethanol(Figure1). Candidayeastscanproduce
mutageniclevelsofACHfromglucoseinvitro[83,86]. Marttilaetal. hasshownthatinlowoxygen
tension,conditionscharacteristicofmultiplenichesintheoralcavity,ACHlevelsproducedinvitro
fromglucosebyCandidacanbeordersofmagnitudelargerthanwhenoxygenispresent[78].
Cancers 2018,10,11 9of23
3.2.3. BiofilmLifestyle—ThePreferredModeofMicrobialGrowthintheGITract
AlongtheGItract,thepreferredlifestyleformicrobesistheformationofbiofilmcommunities.
Biofilmsareorganizedmicrobialcommunitiesattachedtohostsurfacesandencasedinmicrobially
producedextracellularmatrix. Awell-knownexampleofbiofilmsinthehumanbodyisdentalplaque.
In the biofilm mode of growth, microbial metabolism differs from its planktonic counterpart due
to metabolic rewiring [89,90]. Fermentative growth is preferred especially in the deeper niches of
biofilm structures, as oxygen is not as easily available as at the surface. Hypoxic conditions are
commoninmultiplenichesintheoralcavity,suchasgingivalpocketsandthetongue. Duetothese
factors, thefermentativetraitispreferredbyyeastsandbacteriaintheoralcavityandesophagus.
Expression of genes related to ethanol metabolism is upregulated in C. albicans biofilms and high
invitroACHproductionisassociatedwiththebiofilmlifestyleinC.albicans[91]. C.albicansmayaffect
thesite-specificbacterialflorawithinthebiofilmandincreasethenumberofanaerobicbacteriain
thesiteinvitro[92]. ThismayincreasethelocalACHaccumulationduetomicrobialfermentation.
Unfortunately,pastinvitrostudiesonmicrobialACHproductionhasmainlybeenperformedusing
planktoniccells,whichpoorlysimulatesthegrowthandmetabolismofvariousmicrobesinvivo.
3.2.4. TheEffectofAlcoholandSmokingontheMicrobiomeandMicrobiomeEthanolMetabolism
Alcohol consumption, smoking, or both can alter the niche-specific microbiome composition
andcontributetochangesinbiofilmstructuresandthebiofilmmetabolome[93,94]. Interestingly,in
smokers the microbiota has a more potent ability to produce ACH from ethanol both invitro and
invivo[50,79,95,96]. ThismaybedueatleastinparttoselectionofmoreACH-tolerantspecieswithin
thebiofilms. Inalcoholics,yeastovergrowth(inparticularCandida)hasbeenreportedinvivo[97].
AsshowninRussianandJapanesestudypopulations,thegutmicrobiotainalcoholicsdiffersfrom
healthycontrolswithfunctionalchangesinmicrobialethanolmetabolism[98,99]. Itremainstobe
studiedwhethersimilarchangesalsooccurintheupperGItractmicroflora. Thesecompositionaland
functionalchanges,incombinationwithaberrantbarrierfunctionofthegutlinkedtoalcoholuse,can
contributetoincreasedACHexposureandprovideabasisforlocalACH-relatedcarcinogenesis.
3.3. GastricMicrobiome
ThestomachhaslongbeenconsideredasterileenvironmentduetolowpH.However, novel
studies have revealed that a core microbiome exists in a healthy acidic stomach as well [100–102].
The role of pH is critical in terms of balance in the gastric microbiome. A significant microbial
overgrowth and dysbiosis exists in the stomach of patients suffering from hypochlorhydria or
achlorhydria [103,104]. Hypochlorhydria or achlorhydria is usually due to atrophic gastritis or
the use of acid-suppressive medication. Recently, Imhann et al. described a significant increase
ofgeneraubiquitousintheoralmicrobiome(includingStreptococcusandRothia)inthegutofpatients
using acid-suppressive medication [105]. Under anaerobic conditions prevailing in some parts of
a low-acid stomach and in the presence of glucose, bacterial colonization has been reported to
result in an ethanol concentration up to 27 mM (1.3 ) and a maximum ACH concentration of
15.7µM[106,107].Väkeväinenetal.usedculture-depend(cid:104)enttechniquestoidentifythefloraresponsible
for ACH production in hypochlorhydric patients [108]. Various anaerobic and aerobic bacteria as
wellasfungalspecieswereidentified[108].Themicrobiomewasrichintaxaandresembledtheoral
microbiome.ThemostnumerousandprevalentspeciesisolatedwereStreptococcus,Stomatococcus,Neisseria,
andCorynebacteriumspecies.HighmicrobialADHactivitiesandlowK valuesweredeterminedfor
M
multiplespecies,thussupportingthepotentialforhighlocalACHproductionandexposure. More
novelstudiesusingculture-independentmethodshaveshowntheresemblanceofmicrobialflorain
variouscancer-predisposingstagesofstomachandgastriccancerwithoralflora[109,110].
Helicobacterpylori,atrophicgastritis,alcoholconsumption,tobaccosmoking,acid-suppressive
drugs,ALDH2-deficiencyassociatedwithalcoholconsumption,pickledfoods,fermentedsoyfoods,
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and some fermented dairy products are risk factors for stomach cancer [1,2,38,43,111–117]. ACH
isacommonlocaldenominatorofallofthesecancerriskfactorsorconditions. Inthepresenceof
alcohol,theintragastricACHproductioninpatientswithhypochlorhydriaorachlorhydricstomach
secondaryeithertoatrophicgastritisortheuseofacidsuppressivedrugsisincreasedtomutagenic
levels[32,107,108,118,119].
H.pyloriisacorepathogenofthestomach. ThepresenceofH.pylorileadstoadecreaseinthe
diversity of gastric flora. It is the most important etiologic factor for atrophic gastritis and gastric
cancer, and is thus regarded as a group 1 human carcinogen by the IARC [120]. Many H. pylori
strainspossessanactiveADHenzymeandcanproducemarkedlyhighamountsofACHfromethanol
invitro [121,122]. Under low oxygen tension, H. pylori might contribute to local ACH production
through fermentation [123]. However, the invivo ACH-related effects of H. pylori on metaplasia,
dysplasia,andcarcinogenesisarestillunknown.
4. HostMetabolismofEthanolandAcetaldehydeintheUpperGITract
HostmetabolismofethanolandACHmaycontributetomicrobialACHformationfromethanol.
These metabolic pathways are regulated differently by varying organ-specific expression of ADH,
ALDH,andcytochromeP4502E1(CYP2E1)enzymes. Inaddition,genepolymorphismshaveaneffect
ontheactivitiesofenzymesthatmetabolizeethanolandACH.
4.1. OralMucosaandSalivaryGlands
ThehumanoralmucosahasminorADHactivity,10%ofthatoftheesophagusand~6%ofthatin
theliver[54,124]. Therefore,theroleofmucosalADHinACHproductionfromethanolinsalivais
presumablynegligiblecomparedwiththatofmicrobes(seeSection3.2). Buccalmucosaalsoexpresses
CYP2E1[125]. However,thepossiblecontributionofCYP2E1tosalivaryACHconcentrationinthe
presenceofethanolisnotknown. TheoralmucosalacksbothcytoplasmicandmitochondriallowK
M
ALDHenzymes[54]. Thisimportantcharacteristicapparentlyplaysadecisiveroleintheaccumulation
ofACHinsalivaafteralcoholconsumption.
ThereisstrongevidencethatafteralcoholconsumptiontheadditionalACHissecretedinsalivain
ALDH2-deficientindividualsfromthesalivaryglands. Sixtyminutesafteramoderatedose(0.5g/kg)
ofalcohol,threeAsianALDH2-deficientvolunteers(heterozygousforthemutantALDH2*2allele)had
markedlyelevatedACHlevels(3.9–75.0µM)intheirsterileparotidglandsaliva[33]. Insharpcontrast,
therewasnomeasurableACHintheparotidglandsalivafromthreeALDH2-activevolunteers[33].
Furthermore,4-MP,apowerfulinhibitorofthehumanADHenzyme,almostentirelypreventedthe
ethanol-inducedincreaseinsalivaryACHinALDH2-deficientsubjectsbutnotinthosewiththenormal
ALDH2enzyme[34]. Finally,itwasshownthatALDH2deficiencyhasnoeffectonsalivaryACH
concentrationwithoutthepresenceofethanolinthesystemicbloodcirculation[48]. Thesefindings
indicatethatethanolisdiffusedtoparotidglandsfromthebloodcirculationandismetabolizedthere
toACH.However,iftheALDH2enzymeoftheparotidglandsisdeficient,additionalACHissecreted
tosaliva. Itremainstobeestablishedwhethersubmandibularsalivaryglandsplayasimilarrolein
additionalACHsecretioninALDH2-deficientindividuals.
The ADH enzyme encoded by the ADH1C*1 allele metabolizes ethanol to ACH 2.5 times
faster than that encoded by the ADH1C*2 allele. In heavy alcohol drinkers, this polymorphism
isassociatedwithincreasedriskforupperGItractcancer[126]. Inhomozygotes,highersalivaryACH
concentrationswereobservedfollowingalcoholingestionthanthoseinvolunteersheterozygousfor
ADH1CorhomozygousforADH1C*2[127]. AmongJapanesealcoholics,theADH1B*2alleleencoding
thefastethanol-metabolizingADHenzymedoesnothavesignificanteffectonsalivaryACHlevelsin
thepresenceofethanolascomparedtothosewiththeslow-metabolizingADH1B*1/*1genotype[35].
However,slowADHmaybeassociatedwiththeprolongedpresenceofethanolinbloodandsaliva
andthusalsowithextendedexposuretimeoftheupperGItractmucosatomicrobialACH[35].