Review Article Heterocyclic Aromatic Amines Heterocomplexation with Biologically Active Aromatic ...

12 Pages·2014·1.23 MB·English

HindawiPublishingCorporation
ISRNBiophysics
Volume2013,ArticleID740821,11pages
http://dx.doi.org/10.1155/2013/740821
Review Article
Heterocyclic Aromatic Amines Heterocomplexation with
Biologically Active Aromatic Compounds and Its Possible
Role in Chemoprevention
AnnaWoziwodzka,GrzegorzGoBuNski,andJacekPiosik
LaboratoryofBiophysics,IntercollegiateFacultyofBiotechnologyUG-MUG,Kładki24,80-822Gdan´sk,Poland
CorrespondenceshouldbeaddressedtoJacekPiosik;[email protected]
Received29January2013;Accepted24March2013
AcademicEditors:L.Loura,M.P.Ponomarenko,M.Ruiz-Go´mez,andS.Taneva
Copyright©2013AnnaWoziwodzkaetal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Food-borneheterocyclicaromaticamines(HCAs)areknownmutagensandcarcinogenspresentespeciallyinWesternpopulation
diet, which contains large amount of meat and its products. HCAs are capable of interacting with DNA directly through the
formationofcovalentadducts,howeverthisprocessrequiresbiologicalactivationinliver,mainlybycytochromeP450enzymes.
Thisprocessmayproducemutationsandinconsequencemaycontributetothedevelopmentofcancer.However,therearemany
studiesshowingthatseveralbiologicallyactivearomaticcompounds(BACs)mayprotectagainstgenotoxiceffectsofHCAs.Direct
interactionsandnoncovalentheterocomplexesformationmaybeoneofthemostimportantmechanismsofsuchprotection.This
workdescribesseveralBACspresentinhumandiet,whicharecapableofmolecularcomplexesformationwithHCAsandprotect
cellsaswellaswholeorganismsagainstHCAsaction.
1.Introduction above 300∘C by pyrolysis of amino acids and proteins. All
HCAs have at least one aromatic and one heterocyclic ring
Several human mutagens and carcinogens belong to aro- intheirstructure.
matics.Polycyclichydrocarbons(PAHs),aflatoxins,ethidium
HCAs are potent mutagens in the Salmonella/reversion
bromide,acridinedyessuchasICR170andICR191,anticancer
assay as well as proven carcinogens in laboratory ani-
drugs: daunomycin, doxorubicin, and mitoxantrone, and
mals, producing multiorgan tumors, for example, in the
food-derivedheterocyclicaromaticamines(HCAs)canserve
colon,mammaryglands,andprostate[5].Moreover,aclose
as examples. Among them, vast attention is paid to HCAs,
relationship between consumption of thermally processed
which might be responsible for the increase in gastroin-
protein-richfood(mainlymeatandfish)andcanceroccur-
testinal tract cancers, observed particularly in Western diet
rencehasbeenobservedinmanystudies[6–9],indicatinga
populationswithhighmeatintake(Figure1).
significantroleofHCAsinhumancarcinogenesis.
The mutagenic activity of thermally processed meat
ItisbelievedthatHCAsexhibitmutagenic/carcinogenic
was firstly observed in 1939 [1] and subsequently in the
activityonlyaftertheirmetabolicactivation,involvingphase
late 1970s [2]. These observations led to the isolation and
I N-hydroxylation by different isoenzymes of cytochrome
characterization of a new class of potent food-borne muta-
gens/carcinogens,heterocyclicaromaticamines(HCAs)[3]. P450 (mainly CYP1A2) and phase II esterification of the
Basing on their structures, HCAs can be divided into two exocyclic amino group. Products of both phase I and II
classes[4].2-Amino-3-methylimidazo[4,5-f]quinoline(IQ-) activationcanbespontaneouslyconvertedtoarylnitrenium
+
type HCAs are produced during heat processing of crea- ions (R-NH ), capable of forming covalent adducts with
tine/creatinine,sugars,andfreeaminoacidsmixturesintem- DNA[10].
∘
peraturesbelow300 CintheMaillardreaction.Thesecond HCAsareconsideredasimportantfactorsinalifestyle-
class,non-IQ-typeHCAs,areformedathighertemperatures, connected carcinogenesis, according to IARC classified as
2 ISRNBiophysics
NH2 NH2 NH2
N N N
N CH3 N CH3 H3C N CH3
N N CH3 N
IQ MeIQ MeIQx
CH3
CH3 N NH2
N
N N
NH2 N
N N N NH2
H CH3
PhIP Trp-P-2 Glu-P-1
Figure 1: Examples of heterocyclic aromatic amines (HCAs) chemical structures. IQ: 2-amino-3-methylimidazo[4,5-f]quinoline;
MeIQ:2-amino-3,4-dimethylomidazo[4,5-f]quinoline;MeIQx:2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline;PhIP:2-amino-1-methyl-
󸀠 󸀠
6-phenylimidazo[4,5-b]pyridine; Trp-P-2: 3-amino-1-methyl-5H-pyrido[4,3-b]indole; Glu-P-1: 2-amino-6-methyldipyrido[1,2-a: 3,2-
d]imidazole.
2A/Bcarcinogens[11,12],thereforebeingextensivelyinves- 2.MechanismofHeterocomplexation
tigated worldwide with a particular interest in finding
methods to diminish their genotoxic potential. As a result, Mechanism of small aromatic molecules protective action
numerous studies concerning protective effects of several against HCAs activity is often attributed to their ability to
dietary components against HCAs mutagenic and carcino- formheterocomplexesbetweenthesecompounds.Formation
genic activity have been performed. It is estimated that ofsuchcomplexesisdependentonhydrophobicinteractions
approximately600differentindividualcompoundsaswellas between 𝜋-𝜋 electron systems from overlapping aromatic
complex mixtures have been described as antimutagenic or rings present in both molecules. The ability of stacking
anticarcinogenictowardsHCAs,representingdiversemodes heterocomplexes formation can be described by several
of action [13]. Proposed mechanisms of protection involve biophysical parameters, such as enthalpy changes (ΔH) or
inhibition of mutagen formation, inhibition of HCAs acti- association constants (e.g., neighborhood association con-
vating enzymes, induction of HCAs detoxifying enzymes, stant,𝐾AC).Forexample,calculatedvaluesofneighborhood
electrophile-scavenging,enhancedDNArepair,alterationof associationconstants(𝐾AC)forHCAs-theophyllinecomplex
signaling pathways involved in apoptosis and proliferation, formationaregreaterthan𝐾ACvaluesofHCAs-caffeineand
and direct binding of HCAs and their metabolites by inter- HCAs-pentoxifylline heterocomplexes [15]. Andrejuk et al.
ceptormolecules[14]. reported similar relationship between association constant
In thestudies concerningthemolecularmechanismsof values of heterocomplexes formation for several aromatic
chemopreventionagainstHCAsactivityobservedforseveral compounds and theophylline and other methylxanthines
foodcomponents,themainfocusisplacedontheinteraction [16]. This difference was interpreted as a consequence of
of these compounds with different metabolic pathways of a 7-Me group absence in theophylline molecules, which
HCAs, with protective effects resulting from reduction in is present in caffeine and can introduce steric hindrance,
HCAs bioactivation and/or enhancement of their detoxi- reducing heterocomplexation possibility [16]. Possibility of
cation. Nevertheless, taking into consideration that many high-ordercomplexformationcanbeconsideredasanother
natural protective compounds have aromatic moieties in reason for differences between association constants val-
their structure, it seems possible that noncovalent direct ues of stacking complexes formation. Formation of such
interactions, especially stacking (𝜋-𝜋) complexes formation high-order complexes was proposed previously for the
betweenprotectivemoleculesandHCAs,can,atleastinpart, interaction between caffeine (CAF) and anticancer drug
contribute to the observed chemopreventive effects. Such mitoxantrone (MIT) [17]. The authors suggested that the
sequestrationofmutagenmoleculesinheterocomplexeswith formation of MIT-(CAF)2 complex is more probable and
biologically active aromatic compounds (BACs) can reduce morethermodynamicallyfavorablethan1:1heterocomplex,
their bioavailability and in consequence effectively prevent as concluded from lower values of enthalpy changes for
fromtheirgenotoxiceffects(Figure2). 2:1 heterocomplexes [17]. Apart from stacking complexes
This paper summarizes the current state of knowledge formation,directbindingofmutagentoinhibitormolecules
on the importance of heterocomplexation in diminishing can involve electrostatic and H-bond interactions, which
variousbiologicaleffectsofHCAs. can act independently or additionally to heterocomplexes
ISRNBiophysics 3
O OH
H3C CH3 ONa OH
H2C HO O
OH
O
N N
ONa
Cu O OH
N N OH O
H3C ONa OH
O OH
CH3
CH3
CHL EGCG
O
C5H11 O
OH
CH3 CH3
N N O HO O OH
N O
N CH3
H3C O OH O HO CHO
CAF Apigenin Xanthomonasin A
Figure2:Examplesofbiologicallyactivearomaticcompounds(BACs)chemicalstructures.CHL:chlorophyllin;EGCG:(−)-epigallocatechin
gallate;CAF:caffeine.
formation,stabilizing/promoting𝜋-𝜋interactionsbyfacilita- other aromatic compounds protective action is defined as
tionoftwomoleculesdocking.Semenovetal.suggestedthat “protector” mode of action [19]. According to this mech-
stacking heterocomplexes may be stabilized by intermolec- anism, CAF may intercalate to DNA and compete with
ular hydrogen bonds formation, which were demonstrated mutagenforfreebindingsites[21,23].However,“protector”
using vibrational spectroscopy for flavin-mononucleotide modeofCAForotheraromaticcompoundstowardsHCAs
interactionswithethidiumbromideandproflavine[18].All seems unlikely, because biologically activated HCAs form
above-mentionedfactorsmayinfluencetheabilityofseveral covalentadductsratherthanintercalatetoDNA[30,31].
aromaticcompoundstoformstackingheterocomplexeswith
HCAs.
3.RoleofChlorophyllin(CHL)
Therearetwopossiblemechanismsdescribinghowfor-
andOtherPorphyrins
mationofstackingheterocomplexesbetweensmallaromatic
moleculescanmodulatetheirbiologicalactivity.Firstofall,
Chlorophyllin (CHL) is a water-soluble salt of chlorophyll
formation of heterocomplexes between HCAs and several
containingachelatedcopperorsodiumion.CHLisundoubt-
aromatic compounds can decrease concentration of free,
edlyoneofthebest-studiedinterceptormolecules,whichcan
biologically active form of HCAs, thus diminishing their
effectively reduce the activity of several aromatic mutagens
biologicalactivity.Thismechanismofprotectionisknownas
“interceptor”modeofCAFandothermethylxanthinesaction and carcinogens, such as PAHs, aflatoxin B1(AFB1), and
HCAs [32]. Its protective activity is often attributed to
and was proposed by Bedner et al. [19]. The “interceptor”
its ability to form stacking heterocomplexes with planar
modeofCAFwasdescribedandanalyzedforHCAs[15]and
aromaticmutagens,whichcandiminishtheirbioavailability
several other aromatic compounds: ethidium bromide [20,
andthereforeprotectagainsttheirbiologicaleffects.
21],propidiumiodide[19,20],quinacrinemustard,ICR170,
ICR191[22],proflavine[23],andseveralanticancerdrugs[17,
24,25].Similarmechanismwasalsodescribedforinteraction 3.1. Direct Interactions between HCAs and CHL. UV-vis
of chlorophyllin (CHL) [26] and other natural constituents spectroscopy studies demonstrated that CHL forms molec-
of human diet [27] with several aromatic compounds. It ular complexes with IQ with 2:1 stoichiometry and dis-
wasdemonstratedthatCHLformsstackingheterocomplexes sociation constant 𝐾𝑆 = 0.141 ± 0.028mM determined
withHCAsandinconsequencediminishestheirmutagenic from the Benesi-Hildebrand plot [33]. Moreover, CHL was
activity and reduces their absorption from gastrointestinal proven to inhibit mutagenic activity of a broad range of
tract[28,29].ThesecondproposedmechanismofCAFand IQ-type andnon-IQ-typeHCAs in short-termgenotoxicity
4 ISRNBiophysics
assays[29].DirectinteractionsbetweenCHLandHCAswere many environmental and food-borne mutagens, whereas
examined spectrophotometrically, with binding constants Tachino et al. [38] pointed out antioxidant and free rad-
𝐾𝑏 determined from Benesi-Hildebrand plot ranging from ical scavenging effects of CHL and increased mutagen
3.07⋅103M−1to12.74⋅103M−1and2:1and1:1stoichiometries degradation rates in the presence of CHL. These alterna-
ofCHL-mutagencomplexesformationforIQ-typeandnon- tive mechanisms of CHL action can explain its protective
IQ-type HCAs, respectively. Observed protective effects of effects towards nonaromaticcompounds,such as alkylating
CHL against HCAs mutagenic activity were markedly cor- agents:methylmethanesulfonate(MMS),methylnitrosourea
related with strength of mutagen-CHL complex formation, (MNU), ethylnitrosourea (ENU) [39, 40], or radiation-
reflectedbyappropriatebindingconstantvalues.Thesefind- inducedDNAdamage[41,42].Moreover,influenceofCHL
ings support the hypothesis that interception of aromatic on phase II hepatic enzymes was proposed as a mode of
mutagen molecules in noncovalent molecular complexes CHL protective action against low doses of PhIP in rats
with CHL can be considered as an effective mechanism [43].
for CHL protective action, especially for the systems where On the other hand, CHL was demonstrated to protect
interceptormoleculescanreachconcentrationssignificantly againstmutagenicactivityofdirect-actingN-hydroxyHCAs
higherthanpossiblemutagenconcentrations.Furtherstudies derivatives:Trp-P-2[NHOH][44],N-OH-IQ[45],andother
employing molecular modeling analyses of IQ- and non- direct-actingaromaticcompounds:benzo[a]pyrene-7,8-diol-
IQ-type HCA interactions with CHL and its structurally 9,10-epoxide(BPDE)[46]andaflatoxinB1-8,9-epoxide[47].
relatedporphyrinderivativesrevealedthattwofactorscon- In contrary, no inhibitory effect of CHL was observed
󸀠 󸀠
tributetothemolecularcomplexformation[34].Firstly,an against small, nonplanar alkylating agents, N -methyl-N-
exocyclic amine group present in HCA can interact with nitrosourea(MNU)andethylmethanesulfonate(EMS)[46].
acidgroupsonCHLandotherporphyrins.Replacement of Thesefindingsstronglysuggestthatinterceptionofaromatic
an amine group with a nitro group in NO2IQ resulted in mutagensinmolecularcomplexesbyCHLratherthanmodi-
its very weak interaction with CHL (binding constant 𝐾𝑏 ficationofxenobioticmetabolizingenzymesaccountsforthe
estimated at < 0.5⋅103M−1) and was accompanied with a observedantimutageniceffectsofCHL.
lackofantimutagenicactivityofCHLtowardsnitro-IQ[29]. In vitro protective effects of CHL towards DNA were
Secondly, stacking (𝜋-𝜋) interactions between overlapping demonstrated in studies conducted by Pietrzak et al. [26].
aromaticringsiscrucialforstableinteractionsbetweenHCAs Light absorption and fluorescence spectroscopy analyses
andporphyrinderivatives.Thepresenceofmethylgroupsin ofmutagen-CHL-DNAthree-componentmixturesrevealed
chlorophyll derivatives, which can possibly introduce steric thatCHLinducedadose-dependentdecreaseinthemutagen
hindrance and thus interfere with 𝜋-𝜋 interactions, was complexed with DNA concentration. Mutagens used in the
shown to increase the minimal energies of the interactions study,acridineorange,quinacrinemustard,anddoxorubicin,
[34].Atthesametime,theauthorsnoticedthatmanyofthe intercalatetoDNAratherthanformcovalentadducts,which
interceptor molecules, apart from heterocomplexation with istypicalofHCAs.Nevertheless,theseinvitrofindingssup-
aromatic mutagens, are likely to interact with each other, portthehypothesisthatdirectinteractionsbetweenaromatic
forming dimers or longer homoaggregates. This effect can mutagenandCHLmoleculescaninfluenceitsaffinitytobind
complicatethequantitativeanalysisoftheexperimentalsys- toDNA,irrespectivelyofanyothercomplexmechanismsof
tem,asthefractionofthefree/boundinterceptorcannotbe action, which cannot be completely excluded when living
directlyassociatedwiththeamountofaddedmutagen.This organismsareusedasexperimentalmodels.
problemcanbeovercomebyusingthermodynamicalmodels
of mixed aggregation, which allow for infinite homo- and
heterocomplexationbetweeninteractingaromaticmolecules 3.3. Adsorption of HCAs on Insoluble Carriers Containing
and enable one not only to calculate binding constants, CHL. The “interceptor” mode of CHL chemopreventive
that is, neighborhood association constants, but also to actionwasfurtherinvestigatedinstudiesinvolvingadsorp-
determine concentrations of each possible component of tionofaromaticmutagensonCHLboundtoinsolublecarri-
everyanalyzedmutagen-interceptormixture.Suchapproach ers, Sepharose [48] and chitosan [49]. It was demonstrated
wassuccessfullyappliedforinteractionanalysesofHCAs[15] that a broad range of polycyclic planar mutagens, includ-
andotheraromaticmolecules[22,35,36]withcaffeineand ing non-IQ-type HCAs, Trp-P-1, and Trp-P-2, are strongly
othermethylxanthinesandwillbediscussedindetailinthe adsorbed on CHL covalently linked to Sepharose [48].
nextsectionofthispaper. Additionally, the positive relationship between the strength
ofadsorptionofparticularmutagensonCHL-Sepharoseand
the efficacy of CHL in inhibiting their mutagenic activity
3.2. Interception and Other Mechanisms of CHL Action. was revealed [48]. The studies with CHL fixed on chitosan
Although vast majority of studies concerning CHL as a demonstrated that planar molecules having three or more
chemoprotective agent indicate that CHL acts as an “inter- fusedrings(includingIQ,MeIQ,MeIQx,Glu-P-1,andTrp-
ceptor” molecule, forming heterocomplexes with several P-2) efficiently adsorb on CHL-chitosan, whereas for two-
planar mutagens, there are some papers describing dif- ring compounds tested, only those with significant planar
ferent modes of CHL antimutagenic and anticarcinogenic surfaces,suchasPhIP,noncovalentlybindtoCHL-chitosan
activity. Yun et al. [37] suggested that CHL can inactivate [49]. Overall, the adsorption of aromatics to CHL-chitosan
cytochrome P450 (CYP) involved in the bioactivation of wasmuchstrongerthantoCHL-Sepharose,andincontrast
ISRNBiophysics 5
toCHL-Sepharose,mutagensadsorbedtoCHL-chitosanare CHL was proven to protect against PhIP-induced aberrant
difficult to elute even at very low pHs. The adsorption of crypt foci (ACF) in F344 rats, decreasing the uptake of a
HCAs on CHL-chitosan was proven to be relevant for in mutagen from the gastrointestinal tract, as concluded from
vivoprotectiveeffectsofCHL-chitosancoadministeredwith augmented elimination of unmetabolized PhIP in feces in
aromaticcarcinogens.TheamountofTrp-P-2-DNAadducts thepresenceofCHL[57].CHLinhibitedIQ-inducedDNA
in liver and other organs was significantly decreased in a adduct formation in rat liver by interacting with mutagen
micefedwithTrp-P-2andCHL-chitosan[50,51],whichwas directlyandthusreducingitsuptakefromtheintestine[28].
attributedtodiminishedabsorptionofCHL-chitosanbound Inaddition,itwasdemonstratedthatcotreatmentofratswith
mutagen from the gastrointestinal tract. CHL-chitosan was IQandCHLresultedinincreasedurinaryandfecalexcretion
proventobebeneficialinhumans,promotingtheexcretion ofunmetabolizedmutagen,confirmingthatCHLcanactas
ofdioxinsinfecesandsebum[52]. interceptormolecule,interactingwithplanarmutagensand
decreasing their bioavailability in the gastrointestinal tract
and tissues [58]. Studies on Drosophila larvae revealed that
3.4. CHL Influence on Carcinogen Bioavailability. Studies
CHLsignificantlyinhibitedMeIQx-DNAadductformation.
using Caco-2 monolayer transport model have shown that
It was concluded that large doses of CHL can interact with
CHL reduces in vitro carcinogen uptake from the intestine
and effectively intercept MeIQx in feed as well as in the
[53]. In these studies, the influence of CHL on absorption
gastrointestinal tract of larvae, inhibiting its bioavailability
rate of dibenzo[a,l]pyrene (DBP), AFB1, and PhIP through
and absorption [59]. Similar CHL protective effects were
intestinalepithelialcellswasmonitored.CHLwasshownto
alsoobservedtowardsotheraromaticmutagens.Chlorophyll
inhibit the transport of aromatic mutagens, being the most
inhibited absorption of dioxins from the gastrointestinal
effective towards DBP, for which significant inhibition was
tract [60]. Direct interactions between chlorophyll, CHL
observed for 1:10 ratio of mutagen:CHL. For AFB1, 1:100
and, dibenzo[a,l]pyrene (DBP) contributed to the reduced
ratio of mutagen:CHL was sufficient to reduce transport
uptakeofthemutagentotheliver,thusdiminishingstomach
byalmost50%,whereas1:1000ratioofmutagen:CHLwas
and liver cancer incidents in rainbow trout [61]. Chemo-
neededtoinhibitPhIPabsorptionby35%.Thesedifferences
preventive properties of natural chlorophyll and CHL were
in CHL efficiency to inhibit mutagen transport through
also described for another aromatic mutagen/carcinogen-
Caco-2monolayercanbeexplainedbydifferentaffinitiesof
AFB1, and AFB1-induced carcinogenesis in rats [62]. The
CHL for analyzed mutagens, reflected by binding constants
protection resulted from decreased mutagen uptake from
of mutagen-CHL interactions: the highest for BDP-CHL,
the intestines, which was concluded from the increased
intermediate for AFB1-CHL, and, according to Dashwood
excretion of AFB1 in feces of CHL- and chlorophyll-fed
et al. [34], much lower for PhIP-CHL. These findings are
animals.Moreover,molecularcomplexesformationbetween
concordant with the report of Dashwood and Guo [33],
two anticarcinogens and AFB1 were confirmed by fluores-
whosuggested,basingonCHL-mutagenbindingconstants,
cence quenchingexperiments, with1:1 stoichiometriesand
that interceptor CHL action can be effective only when dissociationconstants𝐾𝑑 = 1.22𝜇Mand3.05𝜇MforAFB1-
CHL concentrations are much higher than mutagen con-
chlorophyll and AFB1-CHL interactions, respectively [62].
centrations, for example, IQ:CHL weight ratio of 1:27000
CHLadministeredtogetherwithAFB1inthedietinhibited
ensurescompletesequestrationofIQincomplexeswithCHL.
AFB1-DNA binding in trout. Interestingly, lower protective
Nevertheless, takingintoconsiderationrelativelyhighCHL
effects were observed when AFB1 was given to animals
dosesandlowdietaryexposuretomostcarcinogens,itseems
by intraperitoneal injection, thus significantly reducing the
possible that high CHL:carcinogen ratio could be easily
possibilityofdirectinteractionsbetweenCHLandmutagen
achieved in the gastrointestinal tract. Hence, CHL might
intheintestines[47].SimilarobservationsweremadebyGuo
act as a first line of defense, forming noncovalent hetero-
and Dashwood [63]. They demonstrated that simultaneous
complexeswitharomaticfood-bornemutagens/carcinogens
administrationofIQandCHLtoratsiscrucialtoexertthe
andloweringtheirabsorptionfromthegastrointestinaltract.
mosteffectiveinhibitionofIQ-DNAbinding.Thesefindings
Earlier in vivo studies with rat intestinal loop absorption
serve as additional evidence for interceptor mode of CHL
model gave similar results; CHL administration to isolated
protectiveaction.
intestinalloopsdiminishedthebioavailabilityandabsorption
ofIQ[28].
3.6. Chemoprotective Effects of CHL in Human Studies.
3.5. Animal Studies on CHL Protection towards HCAs and CHLwasproventoactasHCAsprotectiveagentinhuman
Other Dietary Carcinogens. The antigenotoxic potential of studies.ItwasdemonstratedthatCHLtabletsadministeredto
CHLagainstHCAswasobservedinnumerousstudieswith humansexertinhibitoryeffectsagainstDNAdamageinduced
laboratory animals. Cotreatment of F344 rats with PhIP byHCA-containingfriedmeatintargetcolorectalcells[64].
and CHL reduced mammary gland cancer incidences [54]. Clinicalchemopreventiontrialonahumanpopulationwith
SimilarprotectionagainstPhIP-inducedmammarycarcino- high AFB1 exposure revealed that 100mg of dietary CHL
genesis in F344 rats by CHL was observed by Hirose et threetimesperdaysignificantlyreducesthelevelofaflatoxin-
7
al. [55]. Studies on the CHL’s influence on the carcinogenic N -guanine, hepatic DNA adduct biomarker in urine [65].
activity of IQ in rats revealed that CHL protected against ThisstudyclearlydemonstratedthatCHLcanbeaneffective
colon, liver, and small intestine cancers [56]. Furthermore, agent suitable for use in reducing human cancer risk from
6 ISRNBiophysics
dietaryAFB1.Detailedpharmacokineticsstudiesonhuman possibility of direct interactions between planar CAF and
volunteerstreatedwithmicrodoses(30ng)ofAFB1indicated HCAsmoleculeswasproposed[75].
thatchlorophyllaswellasCHLadministrationsignificantly
influencedvariouspharmacokineticparametersandreduced
systemicAFB1absorption[66].Thesefindingsdirectlysug- 4.2. Molecular Complexes Formation between HCAs, CAF,
gest that CHL and chlorophyll exert protective effects in andOtherMethylxanthines. Theideaofheterocomplexation
humansbydecreasingaromaticmutagensbioavailability. between CAF and HCAs was further investigated for IQ-
type HCAs and CAF as well as other methylxanthines
(MTX), pentoxifylline (PTX), and theophylline (TH) [15].
4.RoleofCaffeineandOtherMethylxanthines In order to examine direct interactions between IQ-type
HCAs and MTX, light absorption spectra of mutagens
Caffeine (CAF), a component of several popular beverages titrated with MTX were measured and analyzed for each
(coffee,tea,andenergydrinks),isthemostwidelyconsumed HCA-MTXpair.Distinctivespectralchanges,bathochromic
alkaloid worldwide. Statistical data from the USA revealed and hypochromic shift, were observed, indicating stacking
that the average daily intake of CAF can reach 193mg/day (𝜋-𝜋) complexes formation between analyzed compounds.
per person [67]. Many epidemiological studies suggest that Provided CAF and other MTX form homoaggregates in
caffeine can reduce the risk of cancer [68–71]. Since the aqueoussolution,thedeterminationofassociationconstant
influenceofcaffeineonhumanbodyisexceedinglycomplex, for mutagen-MTX interaction 𝐾𝑏 from Benesi-Hildebrand
itsoverallmechanismofactionasachemopreventiveagent plotisnotaveryaccurateapproach.MTXhomocomplexa-
is still not fully understood. It is commonly known that tioncanbesignificantespeciallyforhigh(milimolarrange)
CAFcanmodulatethemetabolismofxenobiotics,mainlyby MTXconcentrations,hencefree/boundMTXratiocannotbe
theinductionofcytochromeP450enzymes.Therefore,itis directlyrelatedtotheamountofaddedmutagen.Therefore,
believed that CAF may exert its protective action through statistical-thermodynamical model of mixed aggregation
the enhancement of detoxication pathways of numerous [81] which assumes infinite self-aggregation of one of the
carcinogeniccompounds.Ontheotherhand,CAFisinten- interacting ligands was applied in this study in order to
sivelymetabolizedthroughcytochromeP450isoenzyme1A2 calculateneighborhoodassociationconstants𝐾AC ofHCA-
(CYP1A2) [72]. Thus, competitive inhibition of CYP1A2 MTX interactions. The obtained 𝐾AC values of HCA-MTX
2 −1
can be mentioned as one of the possible mechanisms of interactionswerewithin10 M range.Moreover,themodel
CAF protective action towards several procarcinogens, for was used to estimate the concentrations of each possible
example,HCAs,whicharebiologicallyactivatedbyCYP1A2. componentofeveryanalyzedHCA-MTXmixture(i.e.,con-
centration of MTX monomers and MTX homoaggregates
andconcentrationofHCAmonomersandHCA-MTXcom-
4.1.InhibitionofHCAsActivitybyCAF. Itwasdemonstrated plexes).Itwasdemonstratedthat100:1ratioofMTX:HCA
thatcaffeine-richbeverages,suchasdifferenttypesoftea[73– wasenoughtosequestermorethan80%mutagenmolecules
76]aswellascoffee[77],caninhibitHCAsbiologicalactivity. in complexes with MTX [15]. Thus, CAF and other MTX
Moreover,decaffeinatedteawasshowntobelesseffectivein seem to be much moreeffectivein interceptingHCAs than
reducingPhIPmutagenicactivitythanregulartea[75]. CHL,forwhich27000:1CHL:mutagenratiowasproposed
CAFwasproventoattenuatemutageniceffectsofMeIQ, for efficient HCA binding [33]. IQ-MTX binding efficiency
Trp-P-2, MeIQx [78, 79], and PhIP [75]. This protective determined in vitro was confirmed in mutagenicity studies
CAFpotentialwasattributedtotheinhibitionofmicrosomal [15].AmestestexperimentsrevealedthatCAF,PTX,andTH
enzymes (e.g., CYP1A2) involved in HCAs bioactivation. caused a significant dose-dependent reduction of IQ muta-
Experiments with laboratory animals revealed that admin- genicactivity.Astronglinearcorrelationbetweenmutagenic
istration of CAF-containing beverages not only protected activity of IQ-MTX mixtures and free form of IQ suggests
against IQ-induced genotoxicity, but also caused about 20– that CAF and other MTX antimutagenic activities can be
30%inductionofCYP1A2andCYP1A1isoenzymes[76].The explained by sequestration of HCAs molecules in hetero-
lattershouldaugmentratherthandiminishHCAsgenotoxi- complexeswithMTX.Moreover,thesefindingsindicatethat
citybyincreasedHCAsbioactivationratiotohighlyreactive thermodynamicalmodelscanbeausefultooltoestimatethe
arylnitrenium intermediates, suggesting that mechanisms biologicaleffectsofbiologically-activearomaticcompounds
otherthanthemodulationofphaseIenzymescanbeinvolved heterocomplexationandtopredicttowhatextentinterceptor
in observed protective action. The inhibitory effects of tea potential may contribute to overall protective action of
extractstowardspromutagens(IQ,Glu-P-1)aswellasdirect- severalaromaticcompounds.
acting mutagens (9-aminoacridine and N-methyl-N-nitro- Hernaez et al. reported that CAF forms molecular
N-nitrosoguanidine) further support the hypothesis that complexes with direct-acting IQ derivative N-OH-IQ, with
protective effects of CAF cannot be fully explained by its associationconstant𝐾𝑏 < 0.5⋅103M−1,determinedbyspec-
influenceonxenobioticactivationsystem[80].Thepresence trophotometrictitrationexperimentsandBenesi-Hildebrand
of mechanisms additional to the inhibition of cytochrome plot [45]. However, in contrast to the above-mentioned
P450 enzymatic activity was also suggested by Chen and studies [15], no protective effect of CAF against mutagenic
Yen[73].Furthermore,Weisburgeretal.specifiedalternative activityofN-OH-IQwasobservedupto250nmoleCAFper
mechanismsofCAFactionagainstHCAs,amongwhichthe plate.
ISRNBiophysics 7
4.3. Heterocomplexation of MTX with Other Aromatic Lig- required to bind 99.9% is greater than 27000:1) [29, 33].
ands. Althoughthereareonlyfewreportsdescribingstudies ResultsforIQ-MTXinteractionspublishedbyWoziwodzka
oninterceptionroleofCAFandotherMTXtowardsHCAs, et al. demonstrate that MTX reduce mutagenic activity of
heterocomplexationofMTXwithotheraromaticcompounds HCAs despite mixed association constants in the range of
has been broadly investigated. It has been reported previ- 1⋅102M−1 [15]. Considering these data, it can be speculated
ously that CAF and other MTX form stacking aggregates that binding constants in the range of 1⋅103M−1 indicate
witharomaticdyes:acridineorange[82–84],ethidiumbro- the mechanism of HCAs sequestration by polyphenolic
󸀠
mide [20, 81, 85], 4 ,6-diamidino-2-phenylindole (DAPI) compounds as plausible and suggest its role in chemopre-
[81], methylene blue [86], anticancer drugs: doxorubicin, ventiveactionofthesecompoundsagainstHCAsapartfrom
mitoxantrone, and daunomycin [17, 24, 81, 87], antibacte- activation of phase II enzymes and free radicals scaveng-
rial agent norfloxacin [88], vitamin B2 derivative flavin- ing.
mononucleotide [87], neurotoxins [36, 89], and aromatic Additionally, Artemisia argyi analysis performed by
mutagens[22,35].Someofthesereportsrevealinvivoeffects Nakasugi et al. revealed that flavones reduce mutagenic
of observed heterocomplexation, indicating that molecular activityofTrp-P-2[93].ComparisonofAmesmutagenicity
complex formation between aromatic compounds can be testresultsforTrp-P-2anditsactiveform,Trp-P-2[NHOH],
relevantinbiologicalsystems[22,35,36,89]. indicate that, apart from other mechanisms, also hetero-
ItwasdemonstratedthatCAFandotherMTXcaninhibit complexes formation should be considered as important
DNA binding or even induce deintercalation of aromatic chemopreventive mechanism of different flavones, namely
ligands from DNA [20, 21, 84, 88]. It is still discussed eupatilin, jaceosidin, apigenin, and chrysoeriol. Presented
whetherthiseffectresultsbarelyfromthedecreaseinligand results demonstrate that mechanism of heterocomplexes
availability due to its interception by MTX or can be also formation is responsible for up to 30% of overall reduction
contributedtothereductionofbindingsitesonDNAavail- ofHCAsmutagenicactivityintheAmestest.Moreover,pre-
ableforligandscausedbyMTXintercalation.Nevertheless, senteddatademonstrateTrp-P-2fluorescencequenchingin
these findings clearly demonstrate that direct interactions adose-dependentmannerbymentionedflavones,indicating
betweenaromaticcompoundscansignificantlymodifytheir heterocomplexes formation between Trp-P-2 and flavones
biologicalproperties. [93].
Furthermore,Arimoto-Kobayashietal.findingssuggest
that polyphenolic compounds present in beer can act as
5.OtherBACs HCAs interceptors [94]. Beer and other phenol-containing
beverages (i.e., red and white wine, sake, and brandy)
5.1.RoleofPolyphenols. Polyphenolsarenaturallyoccurring,
exhibited strong antimutagenic effects against Trp-P-2 and
aromaticcompoundscontainingatleasttwophenolicrings.
Trp-P-2[NHOH] in the Ames test. The protective effect of
Theyareextensivelystudiedfortheirantimicrobial,anticar-
beer polyphenols was observed only when mutagen was
cinogenic,andantiinflammatoryproperties[90].Inthefield
administered simultaneously with beer. Neither pre- nor
of chemoprevention, they show potential to both scavenge
posttreatmentwithbeercausedanyprotectiveeffectstowards
reactiveoxygenspeciesandformheterocomplexeswitharo-
bacteriaintheAmestest[94].Incontrast,bacteriapretreat-
maticmutagens[91,92].Thelattermechanism,asdescribed
mentwithwinepolyphenoliccompounds(e.g.,ellagicacid)
before, reduces mutagenic potential of aromatic mutagens
resultedindecreaseinTrp-P-2[NHOH]mutagenicactivity,
by formation of molecular complexes with aromatic muta-
probablyduetotheirantioxidativeproperties[95].
gens, thus reducing their bioavailability. Such effects were
described for one of the model mutagens, acridine orange,
andresveratrolbyOsowskietal.[27]. 5.2.RoleofPlantDyes. Plantdyesofaromaticstructurethat
Numerous studies concerning chemopreventive mech- cannot be classified as porphyrins or phenolic compounds
anisms of polyphenolic compounds against heterocyclic also show promising chemopreventive activity. Izawa et
aromatic amines were conducted. Research carried out by al. analyzed Monacus dyes chemopreventive action against
Hernaezetal.revealsthatN-OH-IQ(direct-actingIQderiva- direct-actingTrp-P-2derivative,Trp-P-2[NHOH][96].Their
tive) binds to (−)-epigallocatechin gallate (EGCG), (−)- findings suggest that aromatic dyes present in Monacus
epigallocatechin (EGC), (+)-catechin, and (−)-epicatechin plants, especially red and yellow dye, significantly reduce
gallate (ECG) directly. The binding constants determined HCAsmutagenicactivity,withobservedinhibitoryeffectfor
from Benesi-Hildebrand plot for these polyphenolic com- redandyellowdyesreaching55%.Whatisimportantisthat
pounds are in the range of 1⋅103M−1 [45]. Nevertheless, spectrophotometric study of Trp-P-2[NHOH] interactions
authors of this work suggest that determined interaction withyellowdyeshowedsignificantspectralchangessuggest-
constants are too low to contribute to observed inhibition ingstronginteractionsbetweendyeandmutagen.Moreover,
of HCAs mutagenic activity [45]. However, two works of theauthorssuggestedthatdye-HCAheterocomplexesforma-
Dashwood and Guo demonstrate IQ-CHL interaction con- tion can induce observed degradation of Trp-P-2[NHOH]
stantdeterminedfromBenesi-Hildebrandplotintherange [96]. These results indicate that, apart from chlorophyllin,
of7⋅103M−1.Nonetheless,authorsconsidertheinteractions othernaturaldyescanbealsoinvolvedinprotectiontowards
probableandimportantCHLchemopreventivemechanism, HCAsbyreductionofmutagenbioavailabilityandenhancing
especially for high CHL:IQ ratios (the ratio of CHL:IQ itsdegradationthroughheterocomplexesformation.
8 ISRNBiophysics
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